Compositions and methods of treating cancer with chimeric antigen receptors

ABSTRACT

This disclosure relates to compositions and methods for treating cancer using armored chimeric antigen receptor cells.

BACKGROUND OF DISCLOSURE Field of Invention

This disclosure relates to treatment of cancer using chimeric antigenreceptor T cells.

Technical Background

1. Chimeric antigen receptor T cell therapy

Chimeric antigen receptor (CAR) T cell therapy is a specific form ofcell-based immunotherapy that uses engineered T cells to fight cancer.In CAR T cell therapy, T cells are harvested from a patient's blood,engineered ex vivo to express CARs containing both antigen-binding and Tcell-activating domains, expanded into a larger population, andadministered to the patient. The CAR T cells act as a living drug,binding to cancer cells and bringing about their destruction. Whensuccessful, the effects of CAR T cell treatment tend to be long lasting,as evidenced by detection of CAR T cell persistence and expansion in thepatients long after clinical remission.

2. CAR structure and function

The antigen-binding domain of a CAR is an extracellular region thattargets a surface antigen on tumor cells. Appropriate target antigenscan be proteins, phosphorylated proteins, peptide-MHC, carbohydrates, orglycolipid molecules. Ideal target antigens are widely expressed ontumor cells to enable targeting of a high percentage of the cancercells. Ideal candidate target antigens are also minimally expressed onnormal tissues, limiting off-tumor, on-target toxicity. Theantigen-binding domain of a CAR comprises a targeting moiety, such as anantibody single chain variable fragment (scFv), which is directedagainst the target antigen.

The T cell-activating domain of a CAR is intracellular and activates theT cell in response to the antigen-binding domain interacting with itstarget antigen. A T cell activating domain can contain one or moreco-stimulatory domains, which are the intracellular domains of knownactivating T cell receptors. The selection and positioning ofcostimulatory domains within a CAR construct influence CAR T cellfunction and fate, as costimulatory domains have differential impacts onCAR T cell kinetics, cytotoxic function, and safety profile.

The extracellular antigen-binding and intracellular T cell-activatingdomains of CARs are linked by a transmembrane domain, hinge, andoptionally, a spacer region. The hinge domain is a short peptidefragment that provides conformational freedom to facilitate binding tothe target antigen on the tumor cell. It may be used alone or inconjunction with a spacer domain that projects the scFv away from the Tcell surface. The optimal length of the spacer depends on the proximityof the binding epitope to the cell surface.

CAR T therapy against the B-lymphocyte antigen CD19 (Kymriah©, Novartis)has shown promise in pediatric acute lymphocytic leukemia, and CAR Ttherapy against B-cell maturation antigen (“bb2121,” a Celgene© andbluebirdbio© collaboration) has shown promise againstrelapsed/refractory multiple myeloma. More recent data suggest that theCAR approach can be efficacious against solid tumors. A GD2 CAR naturalkiller T cell (NKT) therapy has shown activity in neuroblastoma (HeczeyA, et al. Invariant NKT cells with chimeric antigen receptor provide anovel platform for safe and effective cancer immunotherapy. Blood;124(18):2824-33, 2014), and mesothelin CAR T with pembrolizumab hasdemonstrated anti-tumor activity in mesothelioma. However, additionaltargets for treating solid tumors are needed.

3. Challenges of CAR T cell therapy

Unfortunately, the complexities of CAR T cell-based therapy can lead toundesirable and unsafe effects. Toxic effects such as neurotoxicity andacute respiratory distress syndrome are potential adverse effects of CART cell therapy and are potentially fatal. Cytokine release syndrome(CRS) is the most common acute toxicity associated with CAR T cells. CRSoccurs when lymphocytes are highly activated and release excessiveamounts of inflammatory cytokines. Serum elevations of interleukin 2,interleukin 6, interleukin 1 beta, GM-CSF, and/or C-reactive protein aresometimes observed in patients with CRS when these factors are assayed.CRS is graded in severity and is diagnosed as one of grades 1-4 (mild tosevere), with more serious cases clinically characterized by high fever,hypotension, hypoxia, and/or multi-organ toxicity in the patient. Onestudy reported that 92% of acute lymphocytic leukemia patients treatedwith an anti-CD19 CAR T cell therapy experienced CRS, and 50% of thesepatients developed grade 3-4 symptoms (Fitzgerald et al., Crit Care Med.45(2): e124-e131 (2017)).

Another challenge to successful CAR T cell immunotherapy isimmunosuppression caused by characteristics of the tumormicroenvironment (TME) of solid tumors. The tumor microenvironment (TME)of solid tumors can be metabolically hostile to CAR T cells due tolimited nutrient availability. This hostility leads to nutrientcompetition, loss of T cell metabolic fitness, and reduced oxygencontent (i.e., “hypoxia”). Hypoxia decreases T cell activation andproliferation, as well as cytokine and lytic enzyme production. A keyplayer in these effects is hypoxia inducible factor-1a (HIF-1α), whichis the alpha subunit of transcription factor hypoxia-inducible factor-1(HIF-1). HIF-1α is a master regulator of cellular and systemichomeostatic response to hypoxia, which make it a key factor in oxygenhomeostasis. HIF-1αalso controls transcription of genes involved inenergy metabolism, angiogenesis, apoptosis, as well as other genes thatfacilitate metabolic adaptation to hypoxia. HIF-1αadditionally plays animportant role in T cell activation. It causes T cells to exhibit a fulleffector phenotype, which includes cytokine and lytic granuleproduction, high glycolysis, high metabolic activity, and high reactiveoxygen species (ROS) production. Such a phenotype leads to low numbersof effector T cells as tumor sites, reduced maintenance of effectorcharacter, and low persistence. It has been shown that a low effectorphenotype leads to increased numbers of effector cells with increasedactivity at tumor sites and greater persistence (Kishton, R. J.,Sukumar, M., & Restifo, N. P. (2017). Metabolic Regulation of T CellLongevity and Function in Tumor Immunotherapy. CellMetab, 26(1),94-109). Therefore, HIF-1α-mediated immunosuppression is also asignificant hurdle that must be overcome to obtain effective andpersistent CAR T cell therapy for solid tumors.

4. Armoring

A recent approach to making CAR T cells that are more resistant totumor-associated immunosuppression is called “armoring.” Armoring is themolecular manipulation of a CAR T cell to express one or more “armoringmolecules” that can counter immunosuppression. For example, expressionof a dominant-negative HIF-1α(HIFlaDN) armoring molecule in pancreaticcancer cells resistant to apoptosis induced by hypoxia and glucosedeprivation due to HIF-1α, rendered the cells sensitive to apoptosis andgrowth inhibition induced by hypoxia and glucose deprivation. (Che etal., Dominant-Negative Hypoxia-Inducible Factor-1a ReducesTumorigenicity of Pancreatic Cancer Cells through the Suppression ofGlucose Metabolism, Am JPathol, 162(4), 1283-1291 (2003)).

Therefore, additional CAR T cell therapies are needed to augment thearmamentarium of effective cancer treatments. Such therapies shouldinclude CAR T cells that effectively treat cancer while minimizing therisk of developing dangerous inflammatory responses, such as CRS. Inaddition, such therapies should include CAR T cells that can persist inthe immunosuppressive TME of solid tumors.

BRIEF SUMMARY OF THE INVENTION

This disclosure describes compositions and methods for using CAR T cellsto treat cancer. As described below, in a first aspect, an isolatednucleic acid sequence encoding (a) a chimeric antigen receptor (CAR),wherein the CAR comprises an antigen-binding domain specific for a cellsurface antigen; and (b) an armoring molecule, wherein the armoringmolecule counters immunosuppression of a cell in a tumormicroenvironment when expressed on a surface of the cell.

In another aspect, the discolure describes a cell comprising a nucleicacid sequence encoding a chimeric antigen receptor (CAR), and a HIFlaDNarmoring molecule expressed on a surface of the cell.

In a further aspect, the discolure describes a cell, comprising: ananti-GPC3 chimeric antigen receptor (CAR) comprising an antigen bindingdomain, wherein the antigen binding domain comprises an antibody, Fab,or an scFv comprising a heavy chain variable region (VH) and a lightchain variable region (VL), wherein the VH comprises a CDR1 comprisingthe amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the aminoacid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acidsequence of SEQ ID NO: 39, and wherein the VL comprises a CDR1comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, aCDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO:44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 orSEQ ID NO: 45; and a HIFIlaDN armoring molecule.

In yet another aspect, the discolure describes a method of treatingcancer, comprising: administering to a subject in need thereof a cell,wherein the cell comprises (a) an chimeric antigen receptor (CAR)specific for a cell surface antigen, and (b) an armoring molecule,wherein the armoring molecule counters immunosuppression of the cell ina tumor microenvironment of the cancer.

These and other features and advantages of the present invention will bemore fully understood from the following detailed description takentogether with the accompanying claims. It is noted that the scope of theclaims is defined by the recitations therein and not by the specificdiscussion of features and advantages set forth in the presentdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a furtherunderstanding of the methods and compositions of the disclosure. Thedrawings illustrate one or more embodiment(s) of the disclosure andtogether with the description serve to explain the principles andoperation of the disclosure.

FIG. 1 . Armoring of GPC3 CAR-T with HIF1aDN. A schematic view of HIF1aDN structure.

FIGS. 2A-2C. HIF1aDN is overexpressed versus endogenous HIFl1.Untransduced (UT) cells, unarmored GPC3 CAR T cells, and GPC3 HIFlaDNCAR T cells were expanded under normoxic (20% O₂) or hypoxic (1% O₂)conditions. 2A. Graph shows the expression of endogenous HIF1α mRNA foreach sample expressed as fold induction over UT expanded at (20% O₂).2B. Endogenous HIF 1 a protein expression in untransduced, unarmoredGPC3 CAR T cells, and GPC3 HIFlaDN CAR T cells at normoxia (20% O₂) andhypoxia (1% 02). β-actin was used as a loading control. 2C. Foldinduction of HIF 1a DN over endogenous HIF1ca mRNA expression inuntransduced (UT), GPC3 CAR T cells, and GPC3 HIF1caDN CAR T cellsexpanded under normoxia (20% O₂) and hypoxia (1% O₂).

FIG. 3 . CAR-T expressing HIF1aDN are less differentiated. Untransduced(UT), GPC3, and GPC3 HIF1aDN CAR T cell differentiation under normoxicor hypoxic conditions were analyzed by flow cytometry. Bar graphrepresents the frequency of CD62LI° ^(w) CD45RO^(high) effector memory Tcells (T_(EM)) evaluated by flow cytometry.

FIG. 4 . Nanostring analysis of stemness in HIF1aDN CAR T cells.Expression of genes associated with stemness (upper portion arrow) andeffector function (lower portion). Genes are expressed as GPC3 HIF 1aDNover GPC3.

FIG. 5 . Dominant negative HIF1a expressing GPC3-CAR-modified T cellsexhibit enhanced oxidative phosphorylation upon acute activation. (A)Upper panel shows OCR of GPC3-CAR-modified and dominant negative HIFlaGPC3-CAR-modified T cells under basal culture conditions and in responseto the activating CD3/CD28 coated beads and oligomycin. Lower panelshows basal OCR and OCR gain after acute activation of resting T cells.Data are shown as mean values ±SD. (B) Upper panel shows ECAR ofGPC3-CAR-modified and dominant negative HIFla GPC3-CAR-modified T cellsunder basal culture conditions and in response to the activatingCD3/CD28 coated beads and exogenous glucose. Lower panel shows basalECAR and ECAR gain after acute activation of resting T cells. Data areshown as mean values ±SD. (**P<0.01, ***P<0.001; Wilcoxon matched pairssigned rank test).

FIG. 6 . HIF1aDN CAR T cells degranulate upon antigen encounter. Flowcytometric analysis of UT, GPC3, GPC3 HIF1aDN CAR T cells for CD107aexpression, a marker of degranulation and toxicity, in the presence ofthe Hep3B (GPC3+) or SNU182 (GPC3-) cells after expansion of CAR-T cellsunder normoxic (left) and hypoxic (right) culture conditions. CAR-Tcells degranulate in vitro in response to antigen encounter even ifexpanded in hypoxia. HIF 1aDN CAR T cells degranulate efficiently evenif they are less activated and differentiated at rest.

FIG. 7 . HIF1aDN CAR T cells produce less IFNγ and IL-2, but similarTNFα upon antigen encounter. Concentration of IFNγ, IL-2 and TNFa in theculture supernatant of UT, GPC3, and GPC3 HIF1aDN CAR T cells expandedin normoxic or hypoxic conditions, 24 hr following antigen (GPC3)encounter under normoxic conditions.

FIG. 8 Ectopic dominant negative HIFla generates polyfunctional T cellsupon stimulation in low oxygen condition. (A) Representative flow plotsfor IFN-7, IL-2, TNF-α intracellular cytokine staining ofGPC3-CAR-modified and dominant negative HIFla GPC3-CAR-modified T cellsafter stimulation with PMA and ionomycin for 6h. (B) Bar diagramsdepicting the quality of the cytokine response by CD4+ and CD8+ T cellsas determined by the Boolean combination of gates identifying IFN-7*,IL-2+ and TNF-α cells. Numbers on Y-axis indicate cell percentages.

FIG. 9 . HIF1aDN CAR T cells quickly and efficiently kill target cells.Targeting and killing, via cytolysis, of Hep3B target cells byuntransduced (UT) cells, GPC3, and GPC3 HIF1αDN CAR T cells weremeasured under normoxic and hypoxic conditions. xCelligence® real timeimpedance-based killing assay (RTCA) was used to measure % cytolysis ofHep3B cells over time. UT cells had minimal cytolytic effect on targetcells, while both GPC3 and HIF1αDN CART cells had significant cytolyticeffect. Unexpectedly, HIF1αDN CAR T cells exhibited faster killing oftarget cells compared to unarmored CAR T cells under both normoxic andhypoxic conditions. These surprising results demonstrate that armoringCAR T cells with HIF1αDN provides additional advantages to CAR T cellsbeyond resistance to hypoxia and suggest that HIF 1aDN may be useful toimprove efficiency of other CAR T cells irrespective of CAR targetantigen.

FIG. 10 . Rate and efficiency of target cell cytolysis per E:T ratiounder normoxic and hypoxic conditions. Cytolytic ability of UT, GPC3,and GPC3 HIF1αDN CAR T cells expanded under normoxic (left) and hypoxic(right) conditions against Hep3B cells at different effector to targetratio (E: T). Values are measured with xCelligence® real timeimpedance-based killing assay (RTCA) and expressed as KT80 (timerequired to kill 80% of the target).

FIG. 11 . Visualization of Hep3B target cell cytolysis. Photomicrographsof Hep3B target cell cytolysis by UT, GPC3, and GPC3 HIF1αDN CAR T cellsat 3 hours post introduction of cells (lower panels) in normoxicconditions. Upper panels show T cells in the absence of target cells.

FIG. 12 . Cytolysis of target cells. Cytolysis of HUH7 and PLC-PRF15target cells by UT, GPC3, and GPC3 HIF1aDN CAR T cells under normoxicconditions with an E:T of 1:1. GPC3 and GPC3 HIF1αDN CAR T cellsexhibited similar rates of cytolysis for each cell type.

FIG. 13 . Cellular proliferation in the presence of target antigen.Antigen-dependent cell proliferation measured by Carboxyfluoresceindiacetate succinimidyl ester (CFSE) dilutions.

FIG. 14 . Expression of HIF1aDN enhances CAR-T efficacy in vivo (HUH7)NSG mice were inoculated with HUH7 cells subcutaneously and, when tumorvolume reached approximately 150 mm³, infused i.v. with 7×10⁶ UT, GPC3,or GPC3 HIF1aDN CAR T cells. Tumor volume has been measured twice aweek.

FIG. 15 . Expression of HIF1aDN enhances CAR-T efficacy in vivo (Hep3B).NSG mice were inoculated with Hep3B cells subcutaneously and, when tumorvolume reached approximately 150 mm³, infused i.v. with 7×10⁶ UT, GPC3,or GPC3 HIF1aDN CAR T cells. Tumor volume has been measured twice aweek.

FIG. 16 . Expression of HIF1aDN increases CAR-T ability to infiltratesolid tumors. NSG mice were inoculated with Hep3B cells subcutaneouslyand, when tumor volume reached approximately 175 mm³, infused i.v. with7×10⁶ UT, GPC3, or GPC3 HIF1αDN CAR T cells. The frequency and number ofT cells in the tumor were evaluated 7 days after infusion by flowcytometry.

FIG. 17 . High level of IFNγ in the serum of mice treated with HIF1aDNCAR-T. Concentration of IFN-γ detected in the serum of mice bearingHep3B tumors and infused with untransduced T cells, unarmored or armoredGPC3 CAR-T cells.

FIG. 18 . HIF1αDN CAR-T maintain a less differentiated phenotype in theperiphery. Frequency of CD70+CD27⁻ CAR-T cells in the spleen of micebearing Hep3B tumors and infused with untransduced T cells, unarmored orarmored GPC3 CAR-T cells.

DETAILED DESCRIPTION 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton, et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger, et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

As used herein, the terms “comprise” and “include” and variationsthereof (e.g., “comprises,” “comprising,” “includes,” and “including”)will be understood to indicate the inclusion of a stated component,feature, element, or step or group of components, features, elements orsteps but not the exclusion of any other component, feature, element, orstep or group of components, features, elements, or steps. Any of theterms “comprising,” “consisting essentially of,” and “consisting of” maybe replaced with either of the other two terms, while retaining theirordinary meanings.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly indicates otherwise.

Percentages disclosed herein can vary in amount by ±10, 20, or 30% fromvalues disclosed and remain within the scope of the contemplateddisclosure.

Unless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values herein thatare expressed as ranges can assume any specific value or sub-rangewithin the stated ranges in different embodiments of the disclosure, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. The term “about” also includes the exactamount. For example, “about 5%” means “about 5%” and also “₅%.” The term“about” can also refer to +10% of a given value or range of values.Therefore, about 5% also means 4.5% - 5.5%, for example. Unlessotherwise clear from context, all numerical values provided herein aremodified by the term “about.”

As used herein, the terms “or” and “and/or” can describe multiplecomponents in combination or exclusive of one another. For example, “x,y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, andz,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”

As used herein, the term “polypeptide” refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain or chains oftwo or more amino acids. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms.

A “protein” as used herein can refer to a single polypeptide, i.e., asingle amino acid chain as defined above, but can also refer to two ormore polypeptides that are associated, e.g., by disulfide bonds,hydrogen bonds, or hydrophobic interactions, to produce a multimericprotein.

An “isolated” substance, e.g., isolated nucleic acid, is a substancethat is not in its natural milieu, though it is not necessarilypurified. For example, an isolated nucleic acid is a nucleic acid thatis not produced or situated in its native or natural environment, suchas a cell. An isolated substance can have been separated, fractionated,or at least partially purified by any suitable technique.

As used herein, the terms “antibody” and “antigen-binding fragmentthereof” refer to at least the minimal portion of an antibody which iscapable of binding to a specified antigen which the antibody targets,e.g., at least some of the complementarity determining regions (CDRs) ofthe variable domain of a heavy chain (VH) and the variable domain of alight chain (VL) in the context of a typical antibody produced by a Bcell. Antibodies or antigen-binding fragments thereof can be or bederived from polyclonal, monoclonal, human, humanized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFvs), single-chainantibodies, disulfide-linked Fvs (sdFvs), fragments comprising either aVL or VH domain alone or in conjunction with a portion of the oppositedomain (e.g., a whole VL domain and a partial VH domain with one, two,or three CDRs), and fragments produced by a Fab expression library. ScFvmolecules are known in the art and are described, e.g., in U.S. Pat. No.5,892,019. Antibody molecules encompassed by this disclosure can be ofor be derived from any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subclass ofimmunoglobulin molecule.

As used herein, the term “polynucleotide” includes a singular nucleicacid as well as multiple nucleic acids, and refers to an isolatednucleic acid molecule or construct, e.g., messenger RNA (mRNA) orplasmid DNA (pDNA). The term “nucleic acid” includes any nucleic acidtype, such as DNA or RNA.

As used herein, the term “vector” can refer to a nucleic acid moleculeas introduced into a host cell, thereby producing a transformed hostcell. A vector can include nucleic acid sequences that permits it toreplicate in a host cell, such as an origin of replication. A vector canalso include one or more selectable marker gene and other geneticelements known in the art. Specific types of vector envisioned here canbe associated with or incorporated into viruses to facilitate celltransformation.

A “transformed” cell, or a “host” cell, is a cell into which a nucleicacid molecule has been introduced by molecular biology techniques. Alltechniques by which a nucleic acid molecule can be introduced into sucha cell, including transfection with viral vectors, transformation withplasmid vectors, and introduction of naked DNA by electroporation,lipofection, and particle gun acceleration are contemplated herein.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an antigen or target (such as an epitope) to itscognate binding domain (such as a paratope). As used herein, the term“avidity” refers to the overall stability of the complex between apopulation of epitopes and paratopes (i.e., antigens and antigen bindingdomains).

As used herein, the terms “treat,” “treatment,” or “treatment of” whenused in the context of treating cancer refer to reducing diseasepathology, reducing or eliminating disease symptoms, promoting increasedsurvival rates, and/or reducing discomfort. For example, treating canrefer to the ability of a therapy when administered to a subject, toreduce disease symptoms, signs, or causes. Treating also refers tomitigating or decreasing at least one clinical symptom and/or inhibitionor delay in the progression of the condition and/or prevention or delayof the onset of a disease or illness.

As used herein, the terms “subject,” “individual,” or “patient,” referto any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include, forexample, humans, non-human primates, dogs, cats, guinea pigs, rabbits,rats, mice, horses, cattle, bears, and so on.

As used herein, the term an “effective amount” or a “therapeuticallyeffective amount” of an administered therapeutic substance, such as aCAR T cell, is an amount sufficient to carry out a specifically statedor intended purpose, such as treating or treatment of cancer. An“effective amount” can be determined empirically in a routine manner inrelation to the stated purpose.

2. Overview

The present disclosure is directed to compositions and methods fortreating cancer using chimeric antigen receptor (CAR) cell therapy. Moreparticularly, the present disclosure concerns CAR cell therapies inwhich the transformed cells, such as T cells, express CARs that forexample target Glypican-3 (GPC3). The CAR constructs transformed cellsexpressing the constructs, and the therapies utilizing the transformedcells disclosed herein can provide robust cancer treatments withminimized risk of cytokine release syndrome (CRS) or indiscriminatecytokine release in non-GPC3 expressing cells.

Without wishing to be bound by theory, GPC3 is believed to be a viablecancer target across multiple modalities, including bispecific T cellengagers, CAR cells, as well as monoclonal antibodies and antibody-drugconjugates (ADCs). GPC3, an onco-fetal antigen, is a GPI-linked heparinsulfate proteoglycan. GPC3 stabilizes the Wnt-Fzd interaction,stimulating Wnt signaling. GPC3 competes with Patched for Hh binding,relieving Smoothened inhibition, and inducing GPC3 degradation. Bothpathways have been shown to stimulate hepatocellular carcinoma (HCC)growth. And, GPC3 expression levels have been shown to correlate withstage and grade of HCC.

Further, it is believed that GPC3 is a promising target for CAR celltherapy. Therefore, antibodies and CAR constructs derivized from theseantibodies have been developed as described herein.

An additional aspect of the present disclosure includes CAR T cells,such as those targeting GPC3 and others that are armored with HIF1 aDNto protect the CAR T cells against immunosuppression associated with thehypoxic tumor microenvironment (TME), for example, of solid tumors.

3. CAR Construct Design

CAR constructs of the present disclosure can have several components,many of which can be selected based upon a desired or refined functionof the resultant CAR construct. In addition to an antigen-bindingdomain, CAR constructs can have a spacer domain, a hinge domain, asignal peptide domain, a transmembrane domain, and one or morecostimulatory domains. Selection of one component over another (i.e.,selection of a specific co-stimulatory domain from one receptor versus aco-stimulatory domain from a different receptor) can influence clinicalefficacy and safety profiles.

4. Antigen Binding Domain

Antigen binding domains contemplated herein can include antibodies orone or more antigen-binding fragments thereof. One contemplated CARconstruct targeting GPC3 comprises a single chain variable fragment(scFv) containing light and heavy chain variable regions from one ormore antibodies specific for GPC3 that are either directly linkedtogether or linked together via a flexible linker (e.g., a repeat ofGGGGS having 1, 2, 3 or more repeats).

The antigen-binding domain of a CAR as disclosed herein can vary in itsbinding affinity for the target protein. The relationship betweenbinding affinity and efficacy can be more nuanced in the context of CARsas compared with antibodies, for which higher affinity is typicallydesirable. For example, preclinical studies on a receptor tyrosinekinase-like orphan receptor 1 (ROR1)-CAR derived from a high-affinityscFv (with a dissociation constant of 0.56 nM) resulted in an increasedtherapeutic index when compared with a lower-affinity variant.Conversely, other examples have been reported that engineering the scFvfor lower affinity improves the discrimination among cells with varyingantigen density. This could be useful for improving the therapeuticspecificity for antigens differentially expressed on tumor versus normaltissues.

A variety of methods can be used to ascertain the binding affinity ofthe antigen-binding domain. In some embodiments, methodologies thatexclude avidity effects can be used. Avidity effects involve multipleantigen-binding sites simultaneously interacting with multiple targetepitopes, often in multimerized structures. Thus, avidity functionallyrepresents the accumulated strength of multiple interactions. An exampleof a methodology that excludes avidity effects is any approach in whichone or both of the interacting proteins is monomeric/monovalent sincemultiple simultaneous interactions are not possible if one or bothpartners contain only a single interaction site.

5. Spacer Domain

A CAR construct of the present disclosure can have a spacer domain toprovide conformational freedom to facilitate binding to the targetantigen on the target cell. The optimal length of a spacer domain maydepend on the proximity of the binding epitope to the target cellsurface. For example, proximal epitopes can require longer spacers anddistal epitopes can require shorter ones. Besides promoting binding ofthe CAR to the target antigen, achieving an optimal distance between aCAR cell and a cancer cell may also help to occlude sterically largeinhibitory molecules from the immunological synapse formed between theCAR cell and the target cancer cell. A CAR can have a long spacer, anintermediate spacer, or a shorter spacer. Long spacers can include aCH2CH3 domain (-220 amino acids) of immunoglobulin G1 (IgGI) or IgG4(either native or with modifications common in therapeutic antibodies,such as a S228P mutation), whereas the CH3 region can be used on its ownto construct an intermediate spacer (-120 amino acids). Shorter spacerscan be derived from segments (<60 amino acids) of CD28, CD8a, CD3 orCD4. Short spacers can also be derived from the hinge regions of IgGmolecules. These hinge regions may be derived from any IgG isotype andmay or may not contain mutations common in therapeutic antibodies suchas the S228P mutation mentioned above.

6. Hinge Domain

A CAR can also have a hinge domain. The flexible hinge domain is a shortpeptide fragment that provides conformational freedom to facilitatebinding to the target antigen on the tumor cell. It may be used alone orin conjunction with a spacer sequence. The terms “hinge” and “spacer”are often used interchangeably—for example, IgG4 sequences can beconsidered both “hinge” and “spacer” sequences (i.e., hinge/spacersequences).

A CAR can further include a sequence comprising a signal peptide. Signalpeptides function to prompt a cell to translocate the CAR to thecellular membrane. Examples include an IgG1 heavy chain signalpolypeptide, Ig kappa or lambda light chain signal peptides,granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2 orCSFR2) signal peptide, a CD8a signal polypeptide, or a CD33 signalpeptide.

7. Transmembrane Domain

A CAR can further include a sequence comprising a transmembrane domain.The transmembrane domain can include a hydrophobic a helix that spansthe cell membrane. The properties of the transmembrane domain have notbeen as meticulously studied as other aspects of CAR constructs, butthey can potentially affect CAR expression and association withendogenous membrane proteins. Transmembrane domains can be derived, forexample, from CD4, CD8α, or CD28.

8. Costimulatory Domain

A CAR can further include one or more sequences that form aco-stimulatory domain. A co-stimulatory domain is a domain capable ofpotentiating or modulating the response of immune effector cells.Co-stimulatory domains can include sequences, for example, from one ormore of CD3zeta (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2,IL-2RP and MyD88/CD40. The choice of co-stimulatory domain influencesthe phenotype and metabolic signature of CAR cells. For example, CD28co-stimulation yields a potent, yet short-lived, effector-likephenotype, with high levels of cytolytic capacity, interleukin-2 (IL-2)secretion, and glycolysis. By contrast, T cells modified with CARsbearing 4-1BB costimulatory domains tend to expand and persist longer invivo, have increased oxidative metabolism, are less prone to exhaustion,and have an increased capacity to generate central memory T cells.

9. Cells

CAR-based cell therapies can be used with a variety of cell types, suchas lymphocytes. Particular types of cells that can be used include Tcells, Natural Killer (NK) cells, Natural Killer T (NKT) cells,Invariant Natural Killer T (iNKT) cells, alpha beta T cells, gamma deltaT cells, viral-specific T (VST) cells, cytotoxic T lymphocytes (CTLs),and regulatory T cells (Tregs). In one embodiment, CAR cells fortreating a subject are autologous. In other embodiments, the CAR cellsmay be from a genetically similar, but non-identical donor (allogeneic).

10. CAR Cell Production

CAR constructs of the present disclosure can include some combination ofthe modular components described herein. For example, in someembodiments of the present disclosure, a CAR construct comprises a GPC3scFv antigen-binding domain. In some embodiments, a CAR comprises aGPC3-2 scFv antigen-binding domain. In some embodiments of the presentdisclosure, a CAR construct comprises a CSFR2 signal peptide. In someembodiments, a CAR construct comprises an IgG4P hinge/spacer domaincarrying an S228P mutation. In some embodiments, a CAR constructcomprises a CD28 transmembrane.

Different co-stimulatory domains can be utilized is the CAR constructsof the present disclosure. In some embodiments, a CAR constructcomprises a co-stimulatory domain from the intracellular domain of CD3z.In some embodiments, a CAR construct comprises a CD28 co-stimulatorydomain. In some embodiments, a CAR construct comprises a 4-1BBco-stimulatory domain. In some embodiments, a CAR construct comprisesco-stimulatory domains from CD3z and CD28. In some embodiments, a CARconstruct comprises co-stimulatory domains from CD3z and 4-1BB. In someembodiments, a CAR construct comprises co-stimulatory domains from allof CD3z, CD28, and 4-1BB. In some embodiments, a CAR construct comprisesco-stimulatory domains from ICOS, OX-40, and/or GITR.

11. CAR Construct Assessment

Constructs of the present disclosure were compared and assessed based onsafety as well as persistence and establishment of central memory. Thelower affinity (high off-rate) scFv, GPC3, was assessed favorably onaccount of its improved safety. The 4-1BB and CD3z co-stimulatorydomains (both in the same construct) were assessed favorably based ontheir contribution to improved persistence and favorable in vivophenotype (more central memory).

12. CAR Embodiments

In some embodiments, the present disclosure provides an isolated nucleicacid sequence encoding a chimeric antigen receptor (CAR) that comprisesan antigen-binding domain specific for a surface antigen on a tumorcell. In some embodiments, the cell surface antigen is a protein, aphosphorylated protein, a peptide-MHC, a carbohydrate, or a glycolipidmolecule.

Examples of contemplated cell surface antigens include CD10, CD16, CD19,CD20, CD22, CD123, CD30, CD34, CD47, CD56, CD80, CD86, CD117, CD133,CD138, CD171, CD37, CD38, CD5, CD7, CD79, 5T4, AFP, AXL, BCMA, B7H3,CDH3, CDH6, CLDN6, CLDN18, CLL-1, CMV, CS1, DLL3, DR5, FBP, GD2, GFRA1,GPA33, GPC3, IL-1-RAP, IL17RA, ITGB7, EBV, ERBB1/EGFR, ERBB2/Her-2,ERBB3, ERBB4, cMet, EGFR vIII, FAP, FOLR1, CEA, CEACAM6, EphA2, HSV-1,HSV-2, HTLV, HPV16-E6, HPV16-E7, IL13Ra2, IgK chain, LGR5, LMP1, LeY,LRP8, MG7, MR1, NRCAM, PMEL, NKG2D ligand, PRAME, PRLR, PVR, ROR1, ROR2,SSX2, STEAPI, STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2,MAGE-A1, MAGE-A3, MAGE-A4, mesothelin (MSLN), MUC1, MUC16, NY-ESO-1,WT1, PDL1, CAIX, CD70, PSMA, and PSCA. Other cell surface antigens arealso contemplated herein.

In some embodiments, the present disclosure provides an isolated nucleicacid sequence encoding a chimeric antigen receptor (CAR) that comprisesan antigen binding domain specific for glypican 3 (GPC3). The antigenbinding domain has an equilibrium dissociation constant (K_(D)) of about100 nanomolar (nM) or less, and the CAR construct does not inducecytokine production in GPC3- cells. In some embodiments, theantigen-binding domain includes an antibody or antigen-binding fragmentthereof. The antigen-binding domain can be a Fab or a single chainvariable fragment (scFv). In some embodiments, the antigen-bindingdomain is an scFv comprising the nucleic acid sequence of SEQ ID NO: 33or SEQ ID NO: 34.

In some embodiments, the CAR further includes a transmembrane domain, acostimulatory domain, and a signal domain. The transmembrane domain canbe a CD28 transmembrane domain. The costimulatory domain can be one ormore of CD3zeta (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2,IL-2RP and MyD88/CD40costimulatory domains. In one specific embodiment,the costimulatory domain is one or more of CD28, 4-1BB, and CD3zetacostimulatory domains. The signal domain can be a sequence encoding aCSFR2 signal peptide.

In some embodiments, the isolated nucleic acid sequence can include ahinge/spacer domain. The hinge/spacer domain can be an IgG4Phinge/spacer.

In some specific embodiments, an isolated nucleic acid sequence encodinga chimeric antigen receptor (CAR) can have the sequence of SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 26.

In other embodiments, the present disclosure provides an anti-GPC3chimeric antigen receptor (CAR) including an antigen-binding domain. Theantigen-binding domain can be an antibody, Fab, or scFv comprising aheavy chain variable region (VH) and a light chain variable region (VL).In some embodiments, the VH can have a CDR1 comprising the amino acidsequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence ofSEQ ID NO: 38, and a CDR3 comprising the amino acid sequence of SEQ IDNO: 39. In some embodiments, the VL can have a CDR1 comprising the aminoacid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising theamino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45.

In some embodiments, the VH can be the amino acid sequence of SEQ ID NO:27 or SEQ ID NO: 29, and the VL can be the amino acid sequence of SEQ IDNO: 28 or SEQ ID NO: 30. In some embodiments, the CAR further can have atransmembrane domain, a costimulatory domain, and a signal domain.

In some specific embodiments, the anti-GPC3 CAR can have the amino acidsequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25.

In other embodiments, the present disclosure provides a vectorcomprising a nucleic acid sequence encoding a chimeric antigen receptor(CAR). The nucleic acid sequence can be SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

In other embodiments, the present disclosure provides a cell comprisinga vector having a nucleic acid sequence of SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ ID NO: 34.

In other embodiments, the present disclosure provides a cell having anucleic acid sequence encoding a chimeric antigen receptor (CAR),wherein the CAR comprises an antigen binding domain specific forglypican 3 (GPC3), wherein the antigen binding domain has an equilibriumdissociation constant (K_(D)) of about 100 nanomolar (nM) or less, andwherein the CAR construct does not induce cytokine production in GPC3-cells. For example, the nucleic acid sequence can be SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 33, or SEQ IDNO: 34.

In other embodiments, the present disclosure provides a cell expressingan anti-GPC3 chimeric antigen receptor (CAR) on an extracellular surfacethereof. The CAR can have an antigen-binding domain that can be anantibody, a Fab, or an scFv each having a heavy chain variable region(VH) and a light chain variable region (VL). The VH can include a CDR1comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 39. The VL can include a CDR1comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, aCDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO:44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 orSEQ ID NO: 45.

In some embodiments, the VH can have the amino acid sequence of SEQ IDNO: 27 or SEQ ID NO: 29. In some embodiments, the VL can have the aminoacid sequence of SEQ ID NO: 28 or SEQ ID NO: 30. The CAR can furtherinclude a transmembrane domain, a costimulatory domain, and a signaldomain. The cell express a CAR having an amino acid sequence of SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 25.

In some embodiments, the present disclosure provides a T cell, a NaturalKiller (NK) cell, a cytotoxic T lymphocyte (CTL), and/or a regulatory Tcell that express a CAR on an extracellular surface thereof, and the CARcan have an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ IDNO: 10, or SEQ ID NO: 25. Such cells can exhibit an anti-tumor immunityupon contacting a tumor cell expressing GPC3.

13. Treatment of Cancers with CARs

In some embodiments, the present disclosure provides CAR cells fortreatment of cancer. The compositions (e.g., antibodies, CAR constructs,and CAR cells) and methods of their use described herein are especiallyuseful for inhibiting neoplastic cell growth or spread. In some aspects,they are particularly useful for inhibiting neoplastic cell growth inwhich GPC3 plays a role.

Neoplasms treatable by the compositions of the disclosure include solidtumors, for example, those of the liver, lung, or ovary. However, thecancers listed herein are not intended to be limiting. For example,types of cancer that are contemplated for treatment herein include, forexample, NSCLC, advanced solid malignancies, biliary tract neoplasms,bladder cancer, colorectal cancer, diffuse large b-cell lymphoma,esophageal neoplasms, esophageal squamous cell carcinoma, extensivestage small cell lung cancer, gastric adenocarcinoma, gastric cancer,gastroesophageal junction cancer, head and neck cancer, head and necksquamous cell carcinoma, hepatocellular carcinoma, Hodgkin lymphoma,lung cancer, melanoma, mesothelioma, metastatic clear cell renalcarcinoma, metastatic melanoma, metastatic non-cutaneous melanoma,multiple myeloma, nasopharyngeal neoplasms, non-Hodgkin lymphoma,ovarian cancer, fallopian tube cancer, peritoneal neoplasms, pleuralmesothelioma, prostatic neoplasms, recurrent or metastatic PD-L1positive or negative SCCHN, recurrent squamous cell lung cancer, renalcell cancer, renal cell carcinoma, SCCHN, hypo pharyngeal squamous cellcarcinoma, laryngeal squamous cell carcinoma, small cell lung cancer,squamous cell carcinoma of the head and neck, squamous cell lungcarcinoma, TNBC, transitional cell carcinoma, unresectable or metastaticmelanoma, urothelial cancer, and urothelial carcinoma.

In one embodiment, cancers contemplated for treatment here include anythat express GPC3 on the cell surfaces of the cancer cells. In onespecific example, cancers contemplated for treatment herein includehepatocellular carcinoma, non-small cell lung cancer, ovarian cancer,and squamous cell lung carcinoma.

14. Armoring

In some embodiments, the present disclosure provides “armored” cells,such as CAR T cells that have one or more genetic modifications thatenhance or optimize cell function by protecting the cell against anenvironmental insult, such as an immunosuppressive cytokine or animmunosuppressive TME. Genetic modifications include, but are notlimited to, enhanced secretion of cytokines, expression of ligands thatinteract with immune cells such as T cell, macrophages, and regulatory Tcells, or an alteration of functional characteristics. One of skill inthe art will understand that armoring a cell, such as a T cell, canprovide many additional benefits not described herein that allow for Tcell survival in the immunosuppressive TME.

In some embodiments, a cell can include a chimeric antigen receptor(CAR) comprising a tumor specific antigen binding domain, wherein theantigen binding domain comprises an antibody, Fab, or an scFv comprisinga heavy chain variable region (VH) and a light chain variable region(VL); and a hypoxia-inducible factor 1α(HIF-1α) dominant negative (HIF1aDN) armoring molecule.

In some embodiments, an armored cell can include a nucleic acid sequenceencoding a chimeric antigen receptor (CAR), wherein the CAR comprises anantigen binding domain specific for glypican 3 (GPC3), wherein theantigen binding domain has an equilibrium dissociation constant (K_(D))of about 100 nanomolar (nM) or less, wherein the CAR construct does notinduce cytokine production in GPC3- cells, and wherein the cellexpresses a HIF1αDN armor molecule.

In some embodiments, an armored cell can include an anti-GPC3 chimericantigen receptor (CAR) comprising an antigen binding domain, wherein theantigen binding domain comprises an antibody, Fab, or an scFv comprisinga heavy chain variable region (VH) and a light chain variable region(VL), wherein the VH comprises a CDR1 comprising the amino acid sequenceof SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ IDNO: 38, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 39,and wherein the VL comprises a CDR1 comprising the amino acid sequenceof SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprising the amino acidsequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3 comprising theamino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45; a HIF1αDNarmoring molecule.

15. Methods of Treatment

CAR-modified cells of the present invention, such as CAR T cells, may beadministered alone or as a pharmaceutical composition with a diluentand/or other components associated with cytokines or cell populations.Briefly, pharmaceutical compositions of the invention can include, forexample, CAR T cells as described herein, with one or morepharmaceutically or physiologically acceptable carrier, diluent, orexcipient. Such compositions can comprise buffers such as neutralbuffered saline, buffered saline, and the like; sulfates; carbohydratessuch as glucose, mannose, sucrose, or dextrans, mannitol; proteins,polypeptides, or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. The pharmaceutical compositions of theinvention may be adapted to the treatment (or prophylaxis).

The CAR-modified cells can also be administered in conjunction with oneor more additional therapies. In one embodiment, the additionaltherapies can include anti-cytokine antibodies. For example, one or moreanti-TNFa antibodies can be used to attenuate toxicity and promoteanti-tumor activity at higher CAR T doses, which can be associated withCRS-like symptoms and weight loss.

The number of CAR cells administered per dose, the number of doses, andfrequency of dosing will depend on various parameters such as thepatient's age, weight, clinical assessment, tumor type, tumor burden,and/or other factors, including the judgment of the attending physician.Any acceptable route of administration is contemplated, such as, withoutlimitation, administration intravenous (e.g., intravenous infusion),parenteral, or subcutaneous routes of administration.

In a particular embodiment, a treatment regimen contemplated can includeone or more biological components, such as a CAR T cell and ananticancer antibody and/or a chemotherapeutic component. For example, itis contemplated that a treatment regimen can additionally include animmune checkpoint inhibitor (ICI), such as those that target thePD-1/PD-L1 axis (PDX) and other immune-oncology (IO) treatments, such asimmune system agonists.

Contemplated antibodies include an anti-PD-L1 antibody such asdurvalumab (MEDI4736), avelumab, atezolizumab, KN035, an anti-PD-1antibody such as nivolumab, pembrolizumab, REGN2810, SHR1210, IBI308,PDR001, Anti-PD-1, BGB-A317, BCD-100, and JS001, and an anti-CTLA4antibody, such as tremelimumab or ipilimumab. Additional antibodies arealso contemplated herein. Any therapeutically effective antibodysubparts are also contemplated herein.

Information regarding durvalumab (or fragments thereof) for use in themethods provided herein can be found in U.S. Pat. Nos. 8,779,108;9,493,565; and 10,400,039 the disclosures of which are incorporatedherein by reference in their entirety. In a specific aspect, durvalumabor an antigen-binding fragment thereof for use in the methods providedherein comprises the variable heavy chain and variable light chain CDRsequences of the 2.14H90PT antibody as disclosed in the aforementionedU.S. patents.

Information regarding tremelimumab (or antigen-binding fragmentsthereof) for use in the methods provided herein can be found in U.S.Pat. No. 6,682,736 (in which tremelimumab is referred to as 11.2.1), thedisclosure of which is incorporated herein by reference in its entirety.

Additional therapeutics (chemotherapies or biologics) contemplatedherein include without limitation cisplatin/gemcitabine or methotrexate,vinblastine, ADRIAMYCIN™ (doxorubicin), cisplatin (MVAC),carboplatin-based regimen, or single-agent taxane or gemcitabine,temozolomide, or dacarbazine, vinflunine, docetaxel, paclitaxel,nab-paclitaxel, Vemurafenib, Erlotinib, Afatinib, Cetuximab,Bevacizumab, Erlotinib, Gefitinib, and/or Pemetrexed. Further examplesinclude drugs targeting DNA damage repair systems, such as poly(ADP-ribose) polymerase 1 (PARP1) inhibitors and therapeutics inhibitingWEEl protein kinase activity, ATR protein kinase activity, ATM proteinkinase activity, Aurora B protein kinase activity, and DNA-PK activity.

Any therapeutic compositions or methods contemplated herein can becombined with one or more of any of the other therapeutic compositionsand methods provided herein.

In some embodiments, the present disclosure provides a method oftreating cancer including administering to a subject in need thereof aneffective amount of a cell comprising an anti-GPC3 chimeric antigenreceptor (CAR) comprising an antigen-binding domain and an armoringmolecule that counters immunosuppression of the cell in a tumormicroenvironment when expressed on a surface of the cell. In anotheraspect, the discolure describes antigen-binding domain can be anantibody, Fab, or scFv comprising a heavy chain variable region (VH) anda light chain variable region (VL). The VH can include a CDR1 comprisingthe amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the aminoacid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acidsequence of SEQ ID NO: 39. The VL can include a CDR1 comprising theamino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, a CDR2 comprisingthe amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 44, and a CDR3comprising the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 45. Insome embodiments, the method further inhibits tumor growth, inducestumor regression, and/or prolongs survival of the subject.

In some embodiments, the armoring molecule is HIF1αDN.

In some embodiments, the cell is an autologous cell. For example, theautologous cell can be selected from the group consisting of a T cell, aNatural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), and aregulatory T cell.

In some embodiments, the cancer treated by the method is a solid tumor.For example, the cancer can be hepatocellular carcinoma, non-small celllung cancer, ovarian cancer, and/or squamous cell lung carcinoma. In aspecific embodiment, the cancer is hepatocellular carcinoma.

It is to be understood that the particular aspects of the specificationare described herein are not limited to specific embodiments presented,and can vary. It also will be understood that the terminology usedherein is for the purpose of describing particular aspects only and,unless specifically defined herein, is not intended to be limiting.Moreover, particular embodiments disclosed herein can be combined withother embodiments disclosed herein, as would be recognized by a skilledperson, without limitation.

Examples

The Examples that follow are illustrative of specific embodiments of thedisclosure, and various uses thereof. They are set forth for explanatorypurposes only and should not be construed as limiting the scope of thedisclosure in any way. A description of terms is provided in Table 1.

TABLE 1 Description of terms Term Description GPC3-1 Anti-GPC3 scFv(lower affinity) GPC3-2 Anti-GPC3 scFv (higher affinity) GPC3-4 Priorart anti-GPC3 scFv GPC3-3 Prior art anti-GPC3 scFv BZ Intracellulardomain of CAR with two co-stimulatory domains of 4-1BB and CD3zeta TZIntracellular domain of CAR with a truncated CD3zeta signaling domain(acting as a signaling incompetent control) 28Z Intracellular domain ofCAR with two co-stimulatory domains of CD28 and CD3zeta 28BZIntracellular domain of CAR with three co-stimulatory domains of CD28,4-1BB, and CD3zeta HIF1αDN Dominant-negative HIF-1α molecule GPC3-1 BZCAR with GPC3 scFv, CSFR2 signal peptide, IgG4P hinge region, and both4-1BB and CD3z co-stimulatory domains GPC3-2 BZ CAR with GPC3-2 scFv,CSFR2 signal peptide, IgG4P hinge region, and both 4-1BB and CD3zco-stimulatory domains aCART-H CAR T cell with GPC3 scFv, CSFR2 signalpeptide, IgG4P hinge region, and both 4-1BB and CD3z co-stimulatorydomains and armored with HIF1αDN CSFR2 Signal peptide used in all CARconstructs IgG4P Hinge sequence used in all CAR constructs Hep3BHepatocellular carcinoma model HUH7 Resistant hepatocellular carcinoma(HCC) model

Example 1: Armoring of GPC3 CAR T Cells with HIFlcaDN Produces LessDifferentiated Cells Summary

In the present example, armoring of GPC3 BZ CAR T cells with a dominantnegative HIF-1α(HIF1αDN) molecule was examined as a potential way toprotect CAR T cells against hypoxia-associated immunosuppression toimprove CAR T effector function and tumor control. Methods

FIG. 1 . HIF1αDN: Dominant-negative HIF-1α molecules were prepared bytruncating the wild-type HIF1α protein at both the N- and C-termini suchthat the protein lacked a DNA-binding domain, oxygen-dependentdegradation domain, and transactivating domains. (Chen J, Zhao 5, NakadaK, et al. Dominant-negative hypoxia-inducible factor-1 alpha reducestumorigenicity of pancreatic cancer cells through the suppression ofglucose metabolism. Am J Pathol. 2003; 162(4):1283-1291.doi:10.1016/s0002-9440(10)63924-7). The truncated HIF1a˜DN sequence (SEQTD NO: 49) corresponds to residues 30-389 of the wild-type HIF1αsequence. Armored CAR T cells: GPC3 BZ CAR T cells were armored withHIF1αDN by expressing HIF1αDN as a C-terminal fusion to GPC3 BZ CAR witha T2A peptide separating the GPC3 BZ CAR and the HIF1αDN. In the HIF1αDNarmored GPC3BZ CAR-T construct, a CD33 signal peptide was used to directsecretion of the CAR and the CAR was modififed with an N-terminal HAepitope tag to facilitate detection of CAR expression on the cellsurface.

FIG. 2 . T cells were left untransduced (UT) or were transduced withGPC3-lor GPC3-1 HIF1αDN constructs and expanded for 11 days undernormoxic (20% O₂) or hypoxic (1% O₂) conditions. FIGS. 2A and 2C. mRNAwas extracted from frozen pellets with RNeasy Mini Kit (Qiagen)following manufacturers' instruction and reverse transcribed to cDNA.Endogenous or dominant negative HIF1α were amplified by Real Time-PCRand expressed as fold induction calculated by the Comparative C(T)Method (Schmittgen TD, Livak KJ. Analyzing real-time PCR data by thecomparative C(T) method. Nat Protoc. 2008; 3(6):1101-1108). FIG. 2B. Theexpression of endogenous HIFla was detected by western blot. Cells wereexpanded as described in FIGS. 2A and 2C and lysed in RIPA buffercontaining proteases and phosphatases inhibitors.

FIG. 3 . The frequency of CD62L^(low)CD45RO^(hi)T effector memory cellswas analyzed by flow cytometry on the surface of UT, GPC3-1 or GPC3 -1HIF1αDN CAR-T cells expanded under normoxic (20% O₂) or hypoxic (1% O₂)7 days after transduction.

FIG. 4 . UT, GPC3-1 or GPC3-1 HIF1αDN CAR-T were and expanded undernormoxic conditions for 11 days. cells mRNA was extracted from frozencell pellets with RNeasy Mini Kit (Qiagen). Gene expression analysis wasperformed with Nanostring CAR-T characterization panel followingmanufacturers' instruction. Graph depicts gene expression as Log2 foldchange over UT.

FIG. 5 . GPC3-CAR modified T-cell products were re-suspended inserum-free unbuffered DMEM medium supplemented with L-glutamine (200 mM)and NaCl (143 mM) for glycolysis stress test or with D-glucose (25 mM),and sodium pyruvate (1 mM) for mitochondrial stress test. Cells werethen plated onto Seahorse cell plates (1×10⁶ cells per well), coatedwith Cell-Tak (Corning) to facilitate T cell attachment. Glycolysisstress test was performed by measuring ECAR (mpH/min) at steady stateand after sequential injection of anti-CD3/CD28 beads (at cell to beadsratio of 1:1) followed by D-glucose (10 mM). Mitochondrial stress testwas performed by measuring OCR (pmol/min) at steady state and aftersequential injection of anti-CD3/CD28 beads (at cell to beads ratio of1:1) followed by oligomycin (0.5 μM). Experiments with the Seahorsesystem utilized the following assay conditions: 2 min mixture; 2 minwait; and 3 min measurement.

Results

Expression of the dominant-negative HIFla was confirmed by Real-TimePCR, while expression of endogenous HIFla was investigated by westernblot. Dominant negative HIFIla was overexpressed compared to theendogenous HIFla. Moreover, expression of the DN did not alter thehypoxia-induced stabilization of the endogenous HIF 1a detected bywestern blot (FIG. 2 ).

T cell fitness and persistence in the tumor microenvironment is relatedto their differentiation status (Kishton, R. J., Sukumar, M., & Restifo,N. P. (2017). Metabolic Regulation of T Cell Longevity and Function inTumor Immunotherapy. CellMetab, 26(1), 94-109.). To investigate theeffect of HIF1αDN expression on T cell differentiation, we analyzed thefrequency of CD62L^(low)CD45RO^(hi)T effector memory cells in GPC3-1 orGPC3-1 HIF1αDN CAR-T after 6 days of normoxic (20% O₂) or hypoxic (1%O₂) expansion. GPC3-1 HIF1αDN maintained a less differentiated phenotypeas judged by the decreased frequency of T_(EM) in both hypoxic andnormoxic consitions compared to unarmored CAR-T (see FIG. 3 ). Notably,cells expanded in hypoxia were more differentiated than cells expandedin normoxia, however the expression of HIF1αDN conferred even in thiscondition a less differentiated phenotype resembling that of UT T cells.

HIF1α is not only a master regulator of the hypoxic response, but also akey intermediate of T cell activation in normoxia and it is associatedto the regulation of multiple signaling and metabolic pathways. To havea comprehensive view of the genetic changes induced by expression ofHIF1αDN, we performed nanostring analysis on purified GPC3-1 or GPC3-1HIF1αDN CAR-T expanded under normoxic (20% O₂) conditions. HIF1αDN CAR Tcells have increased expression of genes associated with stemness anddecreased expression of effector function-associated genes compared tounarmored CAR T cells. Therefore, this result confirms that expressionof HIF1αDN is associated to a less activated and differentiatedphenotype (see FIG. 4 ).

Resting T cells utilize an energy efficient oxidative metabolism butswitch to a highly glycolytic metabolism when stimulated to growth orafter pathogen encounter (Michalek, R. D., & Rathmell, J. C. (2010). Themetabolic life and times of a T-cell. Immunol Rev, 236, 190-202.). Toinvestigate the effect of HIF1αDN expression on the metabolic status ofCAR-T cells, we evaluated glycolysis by analyzing the extracellularacidification rate (ECAR) and mitochondrial oxidative phosphorylation onthe basis of the oxygen consumption rate (OCR), through real-time andlive cell analysis (Seahorse XF) in basal conditions and after antigenexposure. In basal conditions, HIF1αDN CAR T cells exhibited a lower OCRand greater ECAR, indicating that they were more glycolitic. Incontrast, upon antigen exposure, HIF1αDN gained 60% of OCR compared toonly 20% of the unarmored. Conversly, the ECAR gain post-activation forthe unarmored was 340% while only 100% for the armored CAR-T. Thereforeexpression of HIF1αDN rewire cell metabolism and endows CAR-T with theability to respond to increased energy demand under stress (See FIG. 5).

Conclusion

The characteristics of the T cells that are used to mount an anti-cancerimmune response are a critical factor in determining clinical outcome.In particular, treatment of cancer using T cells with characteristics ofheightened cellular longevity is associated with improved anti-tumorresponse (Kishton, R. J., Sukumar, M., & Restifo, N. P. (2017).Metabolic Regulation of T Cell Longevity and Function in TumorImmunotherapy. CellMetab, 26(1), 94-109). Hypoxia and the HIF signalingpathway impact on imune cell fate and function, including theupregulation of glycolytic gene expression. As a result, hypoxia drivescells toward a more differentiated status that is associated to shortpersistence in the tumor and poor outcome. (Kishton, R. J., Sukumar, M.,& Restifo, N. P. (2017). Metabolic Regulation of T Cell Longevity andFunction in Tumor Immunotherapy. CellMetab, 26(1), 94-109.; Krzywinska,E., & Stockmann, C. (2018). Hypoxia, Metabolism and Immune CellFunction. Biomedicines, 6(2)). Our data demonstrate that CAR-Texpressing HIF 1aDN maintain a naive and less activated phenotypecompared to unarmored CAR T cells as shown by analysis of surfacemarkers, gene expression, and mitochondrial function. These observationsindicate that HIF1 aDN CAR T cells are well adapted to withstandimmunosuppressive effects associated with the harsh hypoxic TME of solidtumors.

Example 2 Functional Responses of HIF1αDN CAR T cells Upon TargetExposure Summary

In the present example, degranulation, cytotoxicity, effector cytokineproduction, in vitro killing, and proliferative responses weredetermined in HIFl aDN CAR T cells in association with exposure totarget antigen GPC3.

Methods

FIG. 6 . CAR T Cell Degranulation in Response to GPC3. The indicatedCAR-T cells were co-cultured for 6 hours with Hep3B or A375 in presenceof Golgi Stop and an antibody to the degranulation marker CD107a,directly labeled with a fluorochrome. Target engagement induces CAR-Tdegranulation, and consequent binding of the fluorescently labeledanti-CD107 present in the culture medium. CD107 accumulation detected byflow cytometry is directly proportional to the extent of thedegranulation and indicates lysis of the target cell. 6 hours after theincubation, celle were analyzed by flow cytometry.

FIG. 7 . HIF1αDN CAR T cell Effector Cytokine Production in Response toGPC3: 5×10⁴ CAR-T cells were co-cultured with target cells at a 1:1ratio in RPMI 10% FCS. 24 hours later, supernatants were collected andcytokines analyzed by Meso Scale Discovery 4-plex Kit.

FIG. 8 . GPC3-1 or GPC3-1 HIF1αDN CAR-T were exposed to target cellsexpressing GPC3 in hypoxia or normoxia. Intracellular cytokine stainingof GPC3-CAR-modified and dominant negative HIFla GPC3-CAR-modifiedT-cell was performed after a 6-hour culture in the presence of PMA andionomycin. Cells were then stained with Blue Live/Dead fixable dye andCD3 antibodies followed by fixation and permeabilization with the BDFixation and Permeabilization Buffer (eBioscience) following themanufacturer's protocol. Cells were then subsequently incubated withIFN-7, IL-2 and TNF-α (eBioscience) antibodies for 30 minutes at 4° C.Flow cytometric analysis data were compensated and analyzed with FlowJosoftware (TreeStar). Antibodies used: anti-CD3 (SK7), anti-IL-2(MQ1-17H12), anti- IFN-7 (4S.B3) anti-TNFα (MAb 11) purchased fromeBioscience.

FIG. 9 . CAR-T killing of target expressing cells. Cytotoxity studieswere performed with cellular impedance monitoring technology(xCELLigence). 3×10⁴ Target cells were plated and CAR-T cells culturedunder hypoxic and normoxic conditions were added 24 hours later at theindicated effector: target (E:T) ratio. Percent of cytolysis and KT80were calculated with RTCA software Pro. Images were obtained with RTCAeSight (xCELLigence instrument equipped with live cell imagingcapability). Target cells used: Hep3B: GPC3^(h)ig^(h); HUH7: GPC3^(med);PLC-PRF-15: GPC3^(low); SNU-182 GPC3.

FIG. 10 . Killing Time 80 (KT) was calculated with RDCA software Pro onstudies performed as in FIG. 9 , at the indicated E:T ratio, in normoxiaor hypoxia.

FIG. 11 . Bright field images of real-time monitoring of CAR-T-mediatedcytotoxicity during co-culture with GPC3+ tumor cells. Pictures weretaken after 3 hours of co-culture. Apoptotic features are already clearin the presence of armored CAR-T cells. Upper pictures represent T cellsin the absence of target cells.

FIG. 12 . Percent of cytolysis calculated with RDCA software Pro onCytotoxity studies performed as in FIG. 9 . With the indicated targetcells (HUH7 and PLC/PRF/5).

FIG. 13 . CAR-T were labeled with CFSE following manufacturer'sinstruction and incubated with the indicated cell line. After 3 days thedilution of CFSE was analyzed by flow cytometry.

FIG. 14 . HUH7 cells were implanted in the flank of NSG mice (10mice/group). When tumors reached an average volume of 150 mm³, mice weredosed with 7 million of the indicated CAR-T). Graphs represent tumorvolume measured at the indicated time.

FIG. 15 . Hep3B cells were implanted in the flank of NSG mice (9mice/group). When tumors reached an average volume of 150 mm³, mice weredosed with 7 million of the indicated CAR-T). Graphs represent tumorvolume measured at the indicated time. Results

HIF1αDN CAR T have a less differentiated and activated phenotype,therefore we wanted to test if they were able to mount an effectiveantigen-specific immune response. GPC3-1 CAR-T cells expanded innormoxia degranulated shortly after exposure to GPC3+ Hep3B, and theextent of degranulation was slightly decreased if cells were expandedunder hypoxic conditions. HIF1αDN CAR T cells expanded under normoxicconditions were able to degranulate similarly to unarmored CAR-T;however, there was no reduction of degranulation if expanded underhypoxic conditions. Degranulation was antigen-specific as shown byabsence of CD107 ⁺ cells after incubation with SNU-182. This resultshown that HIF1αDN CAR T can effectively and specifically degranulate inresponse to antigen encounter even if expanded in hypoxia (see FIG. 6 ).

Cytokine secretion is a hallmark of T cell activation; therefore, wewanted to investigate the impact of HIF1αDN expression onantigen-dependent effector cytokine production. HIF1αDN CAR T cellsproduce less IFNγ and IL-2, but similar amounts of TNF-α upon exposureto GPC3 as unarmored CAR T cells under normoxic and hypoxic conditions.These data demonstrate that even if HIF1 aDN CAR T have a lessdifferentiated phenotype they can secrete substantial amount of effectorcytokines in an antigen-dependent way. See FIG. 7 .

T cells that produce multiple cytokines, so-called “polyfunctional” Tcells, provide a more effective immune response than do cells thatproduce only a single cytokine. The frequency of unarmored and armoredCD4 and CD8 T cells producing the three major effector cytokinesIFN-γ/IL-2⁺/TNFa⁺ upon antigen stimulation was similar between unarmoredand armored when cells were exposed to the antigen in normoxicconsitions. Stimulation in hypoxia profoundly decreased the frequency ofunarmored polyfunctional T cells. In contrast, the frequency ofpolyfunctional HIF1αDN CAR-T was only slightly affected by hypoxia (SeeFIG. 8 ) suggesting that expression of the HIF1caDN protects CAR-T fromhypoxia-driven immune-suppression.

Killing of target cells is the most important feature of CAR-T cells;therefore, we analyzed the ability of GPC3 HIF1αDN CAR T to kill targetcells expressing various levels of GPC3. Expression of HIF1αDN isassociated with a less differentiated phenotype; therefore, wehypothesized that HIF1αDN would persist longer than their unarmoredcounterparts but would also kill target cells more slowly. Unexpectedly,HIF1αDN CAR T cells under either normoxic or hypoxic conditions killedHep3B cells faster than unarmored CAR T cells. Hep3B target cells showeda distinct and pronounced pre-apoptotic appearance after only 3 hoursupon exposure to HIF1αDN CAR T (See FIGS. 9-11 ). The ability of GPC3HIF1αDN CAR T to kill other GPC3⁺cell lines with lower GPC3 expression,HUH7 and PLC-PRF15, was similar compared to the unarmored counterpartFIG. 12 . These results demonstrate that GPC3 HIF1αDN CAR T kills cellsexpressing higher level of GPC3 at a faster rate than the unarmored CART.

HIFl a is stabilized in T cells upon antigen recognition and it is animportant mediator of T cell activation, therefore we asked if theexpression of the DN resulted in altered T cell proliferation. CFSElabeled UT, GPC3, and GPC3 HIF1αDN CAR T cells were left unstimulated orco-cultured with Hep3B or HUH7. CFSE is progressively diluted withindaughter cells following each cell division, dilution being proportionalto the extent of proliferation. Flow cytometric analysis after 3 daysshowed that HIF1 aDN CAR T cells proliferated similarly to unmodifiedCAR T cells in response to target cells expressing high and low GPC3.Expression of HIF1 aDN reverts non-specific proliferation of unmodifiedCAR T cells in the absence of antigen, in line with its lessdifferentiated phenotype. This result shows that blocking the HIF 1 apathway with ectopic expression of a dominant negative molecule does notimpair antigen-induced CAR-T cell proliferation (see FIG. 13 ).

Conclusion

HIF1αDN CAR T cells were able to to kill GPC3⁺cancer cells whilemaintaining a less active and more naive phenotype. Moreover, expressionof HIF1αDN partially protected CAR-T cells from hypoxia-induced loss ofpolyfunctionality. Surprisingly, HIF1αDN CAR T cells demonstrated anenhanced ability to kill certain target cells compared to unarmored CART cells in vitro under both normoxic and hypoxic conditions. Thesesurprising results indicate that armoring CAR T cells with HIF1αDN mayprovide improved cytolytic efficacy to CAR T cells in a way unrelated toproviding resistance to hypoxia.

EXAMPLE 3: expression of HIF1αDN improved CAR-T In vivo Efficacy Summary

In the present example, the effectiveness of HIF1αDN armored CAR-T cellsagainst GPC3⁺tumor cells was determined in vivo in two differentxenograft model.

Methods

The hepatocellular carcinoma Huh7 was used to test the in vivoeffectiveness of HIF1αDN armored CAR-T cells in reducing tumor volume.Tumor cells were implanted in the flank of NSG mice (10 mice/group).When tumors reached an average volume of 150 mm³, mice were dosed with 7×10⁶ of the indicated CAR-T or 7 million of untransduced T cells andtumors measured bi-weekly (see FIG. 14 ).

The hepatocellular carcinoma Hep3B was used to test the in vivoeffectiveness of HIF1αDN armored CAR-T cells in reducing tumor volume.Tumor cells were implanted in the flank of NSG mice (10 mice/group).When tumors reached an average volume of 150 mm³, mice were dosed with7×10⁶ of the indicated CAR-T or 7 million of untransduced T cells andtumors measured bi-weekly (see FIG. 15 ).

Ex vivo analysis was performed on Huh7 tumor bearing mice dosed with7×10⁶CAR-T. Four days after infusion tumors were collected from 5 miceper group. The number of CD45+cells was calculated by flow cytometryusing AccuCheck Counting Beads. (See FIG. 16 ).

Mice bearing Hep3B tumors were dosed with 7×10⁶CAR-T cells. For IFNγanalysis, blood was harvested in small volumes seven days after CAR-Tinfusion and serum separated using BD Microtainer Serum Separator Tubes.IFNγ levels were determined using MSD assays (See FIG. 17 ).

Mice bearing Hep3B tumors were dosed with 7×10⁶CAR-T cells. Four daysafter infusion, spleen and tumors were collected and the expression ofCD27 and CD70 was evaluated by flow cytometry staining. (See FIG. 18 ).

Results

GPC3 HIF1αDN CAR-T cells were able to control more effectively HUH7 andinduced Hep3B tumor regression faster than their unarmoured counterparts(see FIGS. 14 and 15 ). We hypothesized that expression of HIF1αDNconfers CAR-T cells with an increased ability to infiltrate hypoxicsolid tumors. Analysis of Hep3B tumors four days after T cell infusionshowed indeed a 2-3 fold higher number of T cells in the HIF1αDN treatedmice, suggesting that the expression of HIF1αDN allows CAR-T cells tobetter infiltrate and proliferate in solid tumors (see FIG. 16 ). Inline with increased number of cells in the tumor, we also detected moreIFN-γ in the serum of mice infused with HIFl aDN armored GPC3 CAR-T.Notably, while in the tumor armored and unarmored CAR-T cells disolayeda CD70+activated phenotype, the HIF1 aDN were less activated in thespleen with na expression. Of CD70 similar to that of UT cells.

Conclusion GPC3 CAR-T expressing HIF1αDN induced faster tumor regressionthan unarmoured GPC3 CAR-T. Tumor regression was associated to asignificant increase in the ability of the cells to infiltrate andproliferate in the tumor. Moreover, although armored cells were fullyactivated in the tumor, they expressed less CD70 than unarmored CAR-T inthe spleen, suggesting that they can maintain a less activated statuseven in vivo. Overall, these observation indicate that blocking theHIF1αpathway by expression of a dominant negative HIFla improve CAR-Tcells in vivo efficacy while preserving a less activated anddifferentiated phenotype.

The embodiments described herein can be practiced in the absence of anyelement or elements, limitation or limitations that are not specificallydisclosed herein. The terms and expressions which have been employed areused as terms of description and not of limitation, and there is nointention that in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the embodiments claimed. Thus, it should be understood thatalthough the present description has been specifically disclosed byembodiments, optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of these embodiments as defined by the description andthe appended claims. Although some aspects of the present disclosure canbe identified herein as particularly advantageous, it is contemplatedthat the present disclosure is not limited to these particular aspectsof the disclosure.

Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The disclosure includes embodiments in whichexactly one member of the group is present in, employed in, or otherwiserelevant to a given product or process. The disclosure includesembodiments in which more than one, or all of the group members arepresent in, employed in, or otherwise relevant to a given product orprocess.

Furthermore, the disclosure encompasses all variations, combinations,and permutations in which one or more limitations, elements, clauses,and descriptive terms from one or more of the listed claims isintroduced into another claim. For example, any claim that is dependenton another claim can be modified to include one or more limitationsfound in any other claim that is dependent on the same base claim. Whereelements are presented as lists, e.g., in Markush group format, eachsubgroup of the elements is also disclosed, and any element(s) can beremoved from the group.

It should it be understood that, in general, where the disclosure, oraspects of the disclosure, is/are referred to as comprising particularelements and/or features, certain embodiments of the disclosure oraspects of the disclosure consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as ifeach independentpatent and publication was specifically and individually indicated to beincorporated by reference. Citation or identification of any referencein any section of this application shall not be construed as anadmission that such reference is available as prior art to the presentinvention.

TABLE 5 Sequences used in the Examples. SEQ ID NO: 1 GPC3 scFv aminoacid sequence SEQ ID NO: 2 GPC3-2 scFv amino acid sequence SEQ ID NO: 3GPC3 BZ CAR amino acid sequence SEQ ID NO: 4 GPC3 TZ CAR amino acidsequence SEQ ID NO: 5 GPC3 28Z CAR amino acid sequence SEQ ID NO: 6 GPC328 BZ CAR amino acid sequence SEQ ID NO: 7 GPC3-2 BZ CAR amino acidsequence SEQ ID NO: 8 GPC3-2 TZ CAR amino acid sequence SEQ ID NO: 9GPC3-2 28Z CAR amino acid sequence SEQ ID NO: 10 GPC3-2 28BZ CAR aminoacid sequence SEQ ID NO: 11 GPC3 BZ CAR nucleic acid sequence SEQ ID NO:12 GPC3 TZ CAR nucleic acid sequence SEQ ID NO: 13 GPC3 28Z CAR nucleicacid sequence SEQ ID NO: 14 GPC3 28BZ CAR nucleic acid sequence SEQ IDNO: 15 GPC3-2 BZ CAR nucleic acid sequence SEQ ID NO: 16 GPC3-2 TZ CARnucleic acid sequence SEQ ID NO: 17 GPC3-2 28Z CAR nucleic acid sequenceSEQ ID NO: 18 GPC3-2 28BZ CAR nucleic acid sequence SEQ ID NO: 19 GPC3-3BZ CAR amino acid sequence SEQ ID NO: 20 GPC3-3 28BZ CAR amino acidsequence SEQ ID NO: 21 GPC3-4 BZ CAR amino acid sequence SEQ ID NO: 22GPC3-3 BZ CAR nucleic acid sequence SEQ ID NO: 23 GPC3-3 28BZ CARnucleic acid sequence SEQ ID NO: 24 GPC3-4 CAR nucleic acid sequence SEQID NO: 25 GPC3 BZ CAR amino acid sequence (WPRE-deleted) SEQ ID NO: 26GPC3 BZ CAR nucleic acid sequence (WPRE-deleted) SEQ ID NO: 27 GPC3 VHSEQ ID NO: 28 GPC3 VL SEQ ID NO: 29 GPC3-2 VH SEQ ID NO: 30 GPC3-2 VLSEQ ID NO: 31 GPC3-3 scFv amino acid sequence SEQ ID NO: 32 GPC3-4 scFvamino acid sequence SEQ ID NO: 33 GPC3 scFv nucleic acid sequence SEQ IDNO: 34 GPC3-2 scFv nucleic acid sequence SEQ ID NO: 35 GPC3-3 scFvnucleic acid sequence SEQ ID NO: 36 GPC3-4 scFv nucleic acid sequenceSEQ ID NO: 37 GPC3 and GPC3-2 VH CDR1 SEQ ID NO: 38 GPC3 and GPC3-2 VHCDR2 SEQ ID NO: 39 GPC3 and GPC3-2 VH CDR3 SEQ ID NO: 40 GPC3 VL CDR1SEQ ID NO: 41 GPC3 VL CDR2 SEQ ID NO: 42 GPC3 VL CDR3 SEQ ID NO: 43GPC3-2 VL CDR1 SEQ ID NO: 44 GPC3-2 VL CDR2 SEQ ID NO: 45 GPC3-2 VL CDR3SEQ ID NO: 46 GPC3 BZ CAR and Dominant-negative HIF-1α nucleic acidsequence SEQ ID NO: 47 GPC3 BZ CAR and Dominant-negative HIF-1α aminoacid sequence

TABLE 6 Sequences SEQ ID NO: 1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKRYFDYWGQGTMVTVSSGGGG SGGGGSGGGGSSYELTQPPSASGTPGQRVTISCSGGSSNIGSNTVNWFRQLPGTAPKLLV YFNNQRPSGVPDRFSGSKSGTSASLAIGGLQSDDEADYYCVAWDDSLNAPVFGGGTKVTV L SEQ ID NO: 2EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCAKGKRYFDYWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVT ISCSGGSSDIGSNTVNWYQQLPGTAPKLLIYYNNQRPSGVPDRFSGSKSGTSASLAISGL QSEDEADYYCATWDDRMYSPVFGGGTKLTV LSEQ ID NO: 3 MLLLVTSLLLCELPHPAFLLIPGVHSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAM SWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGKRYFDYWGQGTMVTVSSGGGGSGGGGSGGGGSSYELTQPPSASGTPGQRVTISCS GGSSNIGSNTVNWFRQLPGTAPKLLVYFNNQRPSGVPDRFSGSKSGTSASLAIGGLQSDD EADYYCVAWDDSLNAPVFGGGTKVTVLESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVA FIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS EIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* SEQ ID NO: 4 MLLLVTSLLLCELPHPAFLLIPGVHSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAM SWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARGKRYFDYWGQGTMVTVSSGGGGSGGGGSGGGGSSYELTQPPSASGTPGQRVTISCS GGSSNIGSNTVNWFRQLPGTAPKLLVYFNNQRPSGVPDRFSGSKSGTSASLAIGGLQSDD EADYYCVAWDDSLNAPVFGGGTKVTVLESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVA FIIFWVRVKFSRSADAPA* SEQ ID NO: 5MLLLVTSLLLCELPHPAFLLIPGVHSEVQL LESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKRYFDYWGQGTMVTVSSGGGGSGGG GSGGGGSSYELTQPPSASGTPGQRVTISCSGGSSNIGSNTVNWFRQLPGTAPKLLVYFNN QRPSGVPDRFSGSKSGTSASLAIGGLQSDDEADYYCVAWDDSLNAPVFGGGTKVTVLESK YGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR* SEQ ID NO: 6MLLLVTSLLLCELPHPAFLLIPGVHSEVQL LESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKRYFDYWGQGTMVTVSSGGGGSGGG GSGGGGSSYELTQPPSASGTPGQRVTISCSGGSSNIGSNTVNWFRQLPGTAPKLLVYFNN QRPSGVPDRFSGSKSGTSASLAIGGLQSDDEADYYCVAWDDSLNAPVFGGGTKVTVLESK YGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* SEQ ID NO: 7 MLLLVTSLLLCELPHPAFLLIPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GKRYFDYWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGGSS DIGSNTVNWYQQLPGTAPKLLIYYNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADY YCATWDDRMYSPVFGGGTKLTVLESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF WVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR*SEQ ID NO: 8 MLLLVTSLLLCELPHPAFLLIPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK GKRYFDYWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGGSS DIGSNTVNWYQQLPGTAPKLLIYYNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADY YCATWDDRMYSPVFGGGTKLTVLESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF WVRVKFSRSADAPA* SEQ ID NO: 9MLLLVTSLLLCELPHPAFLLIPEVQLLESG GGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGKRYFDYWGQGTMVTVSSGGGGSGGGGSGG GGSQSVLTQPPSASGTPGORVTISCSGGSSDIGSNTVNWYQQLPGTAPKLLIYYNNQRPS GVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDRMYSPVFGGGTKLTVLESKYGPP CPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR* SEQ ID NO: 10MLLLVTSLLLCELPHPAFLLIPEVQLLESG GGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCAKGKRYFDYWGQGTMVTVSSGGGGSGGGGSGG GGSQSVLTQPPSASGTPGQRVTISCSGGSSDIGSNTVNWYQQLPGTAPKLLIYYNNQRPS GVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDRMYSPVFGGGTKLTVLESKYGPP CPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* SEQ ID NO: 11 ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAACTGCCCCATCCCGCCTTCCTGCTG ATTCCTGGTGTACACTCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCT GGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATG AGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGT GGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTAT TACTGTGCGAGAGGAAAGCGATACTTTGACTACTGGGGCCAGGGGACAATGGTCACCGTC TCGAGTGGTGGGGGGGGCAGCGGTGGTGGAGGCTCTGGTGGAGGAGGGAGCTCCTATGAG CTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCT GGAGGCAGCTCCAACATCGGAAGTAATACTGTAAACTGGTTCCGGCAGCTCCCAGGAACG GCCCCCAAACTCCTCGTTTATTTTAATAATCAGCGACCCTCAGGGGTCCCTGACCGATTC TCTGGCTCCAAGTCTGGCACCTCGGCCTCCCTGGCCATCGGTGGGCTCCAGTCTGACGAT GAGGCTGACTATTACTGTGTAGCATGGGATGACTCTCTGAATGCTCCGGTGTTCGGCGGA GGGACCAAGGTCACCGTCCTAGAGAGCAAATATGGACCACCATGCCCTCCATGTCCTTTT TGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTATTCTCTGCTGGTCACAGTGGCT TTCATCATCTTCTGGGTCAAGCGAGGCCGGAAGAAACTGCTGTACATCTTCAAACAGCCT TTTATGCGCCCAGTGCAGACAACTCAGGAGGAAGACGGCTGCTCTTGTCGGTTCCCCGAG GAAGAGGAAGGGGGATGTGAGCTGCGCGTGAAGTTTTCTCGAAGTGCCGATGCTCCTGCA TATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAATAC GACGTGCTGGATAAGAGGCGCGGCAGAGACCCAGAAATGGGCGGGAAGCCACGACGGAAA AACCCCCAGGAGGGGCTGTATAATGAACTGCAGAAGGACAAAATGGCCGAGGCTTACAGC GAAATCGGGATGAAGGGAGAGAGAAGGCGCGGAAAAGGCCACGATGGACTGTATCAGGGC CTGAGCACTGCCACCAAGGACACCTACGATGCTCTGCACATGCAGGCACTGCCACCCAGG TGA SEQ ID NO: 12ATGCTGCTGCTGGTGACAAGCCTGCTGCTG TGCGAACTGCCCCATCCCGCCTTCCTGCTGATTCCTGGTGTACACTCCGAGGTGCAGCTG TTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCC TCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG CTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTG AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC AGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGAAAGCGATACTTTGAC TACTGGGGCCAGGGGACAATGGTCACCGTCTCGAGTGGTGGGGGGGGCAGCGGTGGTGGA GGCTCTGGTGGAGGAGGGAGCTCCTATGAGCTGACTCAGCCACCCTCAGCGTCTGGGACC CCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAGGCAGCTCCAACATCGGAAGTAATACT GTAAACTGGTTCCGGCAGCTCCCAGGAACGGCCCCCAAACTCCTCGTTTATTTTAATAAT CAGCGACCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCGGCCTCC CTGGCCATCGGTGGGCTCCAGTCTGACGATGAGGCTGACTATTACTGTGTAGCATGGGAT GACTCTCTGAATGCTCCGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTAGAGAGCAAA TATGGACCACCATGCCCTCCATGTCCTTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTG GCATGTTATTCTCTGCTGGTCACAGTGGCTTTCATCATCTTCTGGGTCCGCGTGAAGTTT TCTCGAAGTGCCGATGCTCCTGCATGA SEQ ID NO: 13ATGCTGCTGCTGGTGACAAGCCTGCTGCTG TGCGAACTGCCCCATCCCGCCTTCCTGCTGATTCCTGGTGTACACTCCGAGGTGCAGCTG TTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCC TCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG CTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTG AAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC AGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGAAAGCGATACTTTGAC TACTGGGGCCAGGGGACAATGGTCACCGTCTCGAGTGGTGGGGGGGGCAGCGGTGGTGGA GGCTCTGGTGGAGGAGGGAGCTCCTATGAGCTGACTCAGCCACCCTCAGCGTCTGGGACC CCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAGGCAGCTCCAACATCGGAAGTAATACT GTAAACTGGTTCCGGCAGCTCCCAGGAACGGCCCCCAAACTCCTCGTTTATTTTAATAAT CAGCGACCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCGGCCTCC CTGGCCATCGGTGGGCTCCAGTCTGACGATGAGGCTGACTATTACTGTGTAGCATGGGAT GACTCTCTGAATGCTCCGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTAGAGAGCAAA TATGGACCACCATGCCCTCCATGTCCTTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTG GCATGTTATTCCCTGCTGGTCACTGTGGCCTTCATCATCTTCTGGGTGCGGAGCAAGCGG AGCCGGCTGCTGCACTCTGACTACATGAACATGACTCCACGGAGACCCGGCCCTACCCGG AAACATTATCAGCCCTACGCCCCACCCAGAGATTTTGCCGCTTATAGGTCCAGGGTGAAG TTTTCTCGCAGTGCAGATGCCCCTGCTTATCAGCAGGGACAGAATCAGCTGTACAACGAG CTGAATCTGGGCAGGCGCGAGGAATACGACGTGCTGGATAAGCGACGGGGCAGAGACCCC GAAATGGGAGGGAAGCCCAGAAGGAAAAACCCTCAGGAGGGGCTGTATAATGAACTGCAG AAGGACAAAATGGCAGAGGCCTACAGTGAAATCGGGATGAAGGGAGAGCGCCGACGGGGA AAAGGCCACGATGGACTGTATCAGGGCCTGTCTACTGCCACCAAGGACACCTACGATGCC CTGCACATGCAGGCTCTGCCTCCACGCTGASEQ ID NO: 14 ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAACTGCCCCATCCCGCCTTCCTGCTG ATTCCTGGTGTACACTCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCT GGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATG AGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGT GGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTAT TACTGTGCGAGAGGAAAGCGATACTTTGACTACTGGGGCCAGGGGACAATGGTCACCGTC TCGAGTGGTGGGGGGGGCAGCGGTGGTGGAGGCTCTGGTGGAGGAGGGAGCTCCTATGAG CTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCT GGAGGCAGCTCCAACATCGGAAGTAATACTGTAAACTGGTTCCGGCAGCTCCCAGGAACG GCCCCCAAACTCCTCGTTTATTTTAATAATCAGCGACCCTCAGGGGTCCCTGACCGATTC TCTGGCTCCAAGTCTGGCACCTCGGCCTCCCTGGCCATCGGTGGGCTCCAGTCTGACGAT GAGGCTGACTATTACTGTGTAGCATGGGATGACTCTCTGAATGCTCCGGTGTTCGGCGGA GGGACCAAGGTCACCGTCCTAGAGAGCAAATATGGACCACCATGCCCTCCATGTCCTTTT TGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTATTCCCTGCTGGTCACTGTGGCC TTCATCATCTTCTGGGTGCGGAGCAAGCGGAGCCGGCTGCTGCACTCTGACTACATGAAC ATGACTCCACGGAGACCCGGCCCTACCCGGAAACATTATCAGCCCTACGCCCCACCCAGA GATTTTGCCGCTTATAGGTCCAAGCGCGGCCGAAAGAAACTGCTGTACATCTTCAAACAG CCCTTCATGAGACCCGTCCAGACAACTCAGGAGGAAGACGGCTGCAGCTGTAGGTTCCCC GAGGAAGAGGAAGGGGGATGTGAGCTGAGGGTGAAGTTTTCTCGCAGTGCAGATGCCCCT GCTTATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAA TACGACGTGCTGGATAAGCGACGGGGCAGAGACCCCGAAATGGGAGGGAAGCCCAGAAGG AAAAACCCTCAGGAGGGGCTGTATAATGAACTGCAGAAGGACAAAATGGCAGAGGCCTAC AGTGAAATCGGGATGAAGGGAGAGCGCCGACGGGGAAAAGGCCACGATGGACTGTATCAG GGCCTGTCTACTGCCACCAAGGACACCTACGATGCCCTGCACATGCAGGCTCTGCCTCCA CGCTGA SEQ ID NO: 15ATGCTGCTGCTGGTGACAAGCCTGCTGCTG TGCGAACTGCCCCATCCCGCCTTCCTGCTGATTCCTGAGGTCCAGCTGCTGGAGAGCGGA GGAGGACTGGTGCAGCCTGGAGGAAGTCTGCGACTGTCATGCGCCGCTAGCGGCTTCACC TTCAGCTCCTATGCAATGAGCTGGGTGCGACAGGCACCAGGCAAGGGGCTGGAGTGGGTC TCCGCTATCTCCGGCTCTGGAGGCTCTACTTACTATGCAGACAGTGTGAAGGGGCGGTTC ACAATCTCCAGAGATAACTCTAAGAACACTCTGTACCTGCAGATGAACTCTCTGAGAGCT GAGGACACCGCAGTGTACTATTGCGCCAAGGGCAAAAGGTACTTTGATTATTGGGGACAG GGCACTATGGTGACCGTCTCTAGTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGA GGAGGCAGTCAGTCAGTGCTGACACAGCCACCTAGCGCCTCCGGAACCCCAGGACAGCGG GTCACAATCTCTTGTAGTGGGGGATCAAGCGACATTGGGAGCAACACCGTGAATTGGTAT CAGCAGCTGCCTGGAACAGCTCCAAAGCTGCTGATCTACTATAACAATCAGAGGCCCTCC GGCGTCCCTGATCGCTTCTCAGGCAGCAAATCCGGGACTTCTGCAAGTCTGGCCATTAGT GGCCTGCAGTCAGAGGACGAAGCCGATTACTATTGTGCTACCTGGGACGATAGGATGTAC TCTCCCGTGTTCGGCGGGGGAACAAAGCTGACTGTCCTGGAGAGCAAATATGGACCACCA TGCCCTCCATGTCCTTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTATTCT CTGCTGGTCACAGTGGCTTTCATCATCTTCTGGGTCAAGCGAGGCCGGAAGAAACTGCTG TACATCTTCAAACAGCCTTTTATGCGCCCAGTGCAGACAACTCAGGAGGAAGACGGCTGC TCTTGTCGGTTCCCCGAGGAAGAGGAAGGGGGATGTGAGCTGCGCGTGAAGTTTTCTCGA AGTGCCGATGCTCCTGCATATCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAATCTG GGCCGGAGAGAGGAATACGACGTGCTGGATAAGAGGCGCGGCAGAGACCCAGAAATGGGC GGGAAGCCACGACGGAAAAACCCCCAGGAGGGGCTGTATAATGAACTGCAGAAGGACAAA ATGGCCGAGGCTTACAGCGAAATCGGGATGAAGGGAGAGAGAAGGCGCGGAAAAGGCCAC GATGGACTGTATCAGGGCCTGAGCACTGCCACCAAGGACACCTACGATGCTCTGCACATG CAGGCACTGCCACCCAGGTGA SEQ ID NO: 16ATGCTGCTGCTGGTGACAAGCCTGCTGCTG TGCGAACTGCCCCATCCCGCCTTCCTGCTGATTCCTGAGGTCCAGCTGCTGGAGAGCGGA GGAGGACTGGTGCAGCCTGGAGGAAGTCTGCGACTGTCATGCGCCGCTAGCGGCTTCACC TTCAGCTCCTATGCAATGAGCTGGGTGCGACAGGCACCAGGCAAGGGGCTGGAGTGGGTC TCCGCTATCTCCGGCTCTGGAGGCTCTACTTACTATGCAGACAGTGTGAAGGGGCGGTTC ACAATCTCCAGAGATAACTCTAAGAACACTCTGTACCTGCAGATGAACTCTCTGAGAGCT GAGGACACCGCAGTGTACTATTGCGCCAAGGGCAAAAGGTACTTTGATTATTGGGGACAG GGCACTATGGTGACCGTCTCTAGTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGA GGAGGCAGTCAGTCAGTGCTGACACAGCCACCTAGCGCCTCCGGAACCCCAGGACAGCGG GTCACAATCTCTTGTAGTGGGGGATCAAGCGACATTGGGAGCAACACCGTGAATTGGTAT CAGCAGCTGCCTGGAACAGCTCCAAAGCTGCTGATCTACTATAACAATCAGAGGCCCTCC GGCGTCCCTGATCGCTTCTCAGGCAGCAAATCCGGGACTTCTGCAAGTCTGGCCATTAGT GGCCTGCAGTCAGAGGACGAAGCCGATTACTATTGTGCTACCTGGGACGATAGGATGTAC TCTCCCGTGTTCGGCGGGGGAACAAAGCTGACTGTCCTGGAGAGCAAATATGGACCACCA TGCCCTCCATGTCCTTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTACTCC CTGCTGGTCACTGTGGCCTTCATCATCTTCTGGGTGCGGGTGAAGTTTTCTCGCAGTGCC GACGCTCCCGCATGA SEQ ID NO: 17ATGCTGCTGCTGGTGACAAGCCTGCTGCTG TGCGAACTGCCCCATCCCGCCTTCCTGCTGATTCCTGAGGTCCAGCTGCTGGAGAGCGGA GGAGGACTGGTGCAGCCTGGAGGAAGTCTGCGACTGTCATGCGCCGCTAGCGGCTTCACC TTCAGCTCCTATGCAATGAGCTGGGTGCGACAGGCACCAGGCAAGGGGCTGGAGTGGGTC TCCGCTATCTCCGGCTCTGGAGGCTCTACTTACTATGCAGACAGTGTGAAGGGGCGGTTC ACAATCTCCAGAGATAACTCTAAGAACACTCTGTACCTGCAGATGAACTCTCTGAGAGCT GAGGACACCGCAGTGTACTATTGCGCCAAGGGCAAAAGGTACTTTGATTATTGGGGACAG GGCACTATGGTGACCGTCTCTAGTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGA GGAGGCAGTCAGTCAGTGCTGACACAGCCACCTAGCGCCTCCGGAACCCCAGGACAGCGG GTCACAATCTCTTGTAGTGGGGGATCAAGCGACATTGGGAGCAACACCGTGAATTGGTAT CAGCAGCTGCCTGGAACAGCTCCAAAGCTGCTGATCTACTATAACAATCAGAGGCCCTCC GGCGTCCCTGATCGCTTCTCAGGCAGCAAATCCGGGACTTCTGCAAGTCTGGCCATTAGT GGCCTGCAGTCAGAGGACGAAGCCGATTACTATTGTGCTACCTGGGACGATAGGATGTAC TCTCCCGTGTTCGGCGGGGGAACAAAGCTGACTGTCCTGGAGAGCAAATATGGACCACCA TGCCCTCCATGTCCTTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTATTCC CTGCTGGTCACAGTGGCCTTCATCATCTTCTGGGTGCGGAGCAAGCGGAGCCGGCTGCTG CACTCTGACTACATGAACATGACCCCCCGGAGACCCGGCCCTACAAGAAAGCATTATCAG CCTTACGCCCCACCCAGGGACTTCGCAGCTTATCGCTCCCGAGTGAAATTTTCTCGCAGT GCAGATGCCCCCGCTTATCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAATCTGGGG AGGCGCGAGGAATACGACGTGCTGGATAAGCGACGGGGCCGGGACCCCGAAATGGGAGGA AAGCCTAGAAGGAAAAACCCACAGGAGGGCCTGTATAATGAACTGCAGAAGGACAAAATG GCAGAGGCCTACAGCGAAATCGGAATGAAGGGAGAGCGCCGACGGGGCAAAGGACACGAT GGCCTGTATCAGGGGCTGAGCACCGCCACAAAGGACACCTACGATGCCCTGCACATGCAG GCTCTGCCTCCACGCTGA SEQ ID NO: 18ATGCTGCTGCTGGTGACAAGCCTGCTGCTG TGCGAACTGCCCCATCCCGCCTTCCTGCTGATTCCTGAGGTCCAGCTGCTGGAGAGCGGA GGAGGACTGGTGCAGCCTGGAGGAAGTCTGCGACTGTCATGCGCCGCTAGCGGCTTCACC TTCAGCTCCTATGCAATGAGCTGGGTGCGACAGGCACCAGGCAAGGGGCTGGAGTGGGTC TCCGCTATCTCCGGCTCTGGAGGCTCTACTTACTATGCAGACAGTGTGAAGGGGCGGTTC ACAATCTCCAGAGATAACTCTAAGAACACTCTGTACCTGCAGATGAACTCTCTGAGAGCT GAGGACACCGCAGTGTACTATTGCGCCAAGGGCAAAAGGTACTTTGATTATTGGGGACAG GGCACTATGGTGACCGTCTCTAGTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGA GGAGGCAGTCAGTCAGTGCTGACACAGCCACCTAGCGCCTCCGGAACCCCAGGACAGCGG GTCACAATCTCTTGTAGTGGGGGATCAAGCGACATTGGGAGCAACACCGTGAATTGGTAT CAGCAGCTGCCTGGAACAGCTCCAAAGCTGCTGATCTACTATAACAATCAGAGGCCCTCC GGCGTCCCTGATCGCTTCTCAGGCAGCAAATCCGGGACTTCTGCAAGTCTGGCCATTAGT GGCCTGCAGTCAGAGGACGAAGCCGATTACTATTGTGCTACCTGGGACGATAGGATGTAC TCTCCCGTGTTCGGCGGGGGAACAAAGCTGACTGTCCTGGAGAGCAAATATGGACCACCA TGCCCTCCATGTCCTTTTTGGGTCCTGGTGGTCGTGGGAGGCGTGCTGGCATGTTATTCC CTGCTGGTCACTGTGGCCTTCATCATCTTCTGGGTGCGGAGCAAGCGGAGCCGGCTGCTG CACTCTGACTACATGAACATGACTCCACGGAGACCCGGCCCTACCCGGAAACATTATCAG CCCTACGCCCCACCCAGAGATTTTGCCGCTTATAGGTCCAAGCGCGGCCGAAAGAAACTG CTGTACATCTTCAAACAGCCCTTCATGAGACCCGTCCAGACAACTCAGGAGGAAGACGGC TGCAGCTGTAGGTTCCCCGAGGAAGAGGAAGGGGGATGTGAGCTGAGGGTGAAGTTTTCT CGCAGTGCAGATGCCCCTGCTTATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAAT CTGGGCAGGCGCGAGGAATACGACGTGCTGGATAAGCGACGGGGCAGAGACCCCGAAATG GGAGGGAAGCCCAGAAGGAAAAACCCTCAGGAGGGGCTGTATAATGAACTGCAGAAGGAC AAAATGGCAGAGGCCTACAGTGAAATCGGGATGAAGGGAGAGCGCCGACGGGGAAAAGGC CACGATGGACTGTATCAGGGCCTGTCTACTGCCACCAAGGACACCTACGATGCCCTGCAC ATGCAGGCTCTGCCTCCACGCTGA SEQ ID NO: 19MLLLVTSLLLCELPHPAFLLIPDVVMTQSP LSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGGSGGGGSQ VQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYS QKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSDKTHTC PPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR* SEQ ID NO: 20MLLLVTSLLLCELPHPAFLLIPDVVMTQSP LSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGGGSGGGGSQ VQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYS QKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSDKTHTC PPCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR* SEQ ID NO: 21 MLLLVTSLLLCELPHPAFLLIPQVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGLHWVR QAPGKGLEWVAAISYDGSKKYYADSVKGRLTISRDNSKNTLYLQMNSLRPDDTALYFCAR GWFVEPLSWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGORVTISCSGSS SNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEAD YYCAAWDDSLNGYVFGTGTKLTVLESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFII FWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR*SEQ ID NO: 22 ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAACTGCCCCATCCCGCCTTCCTGCTG ATTCCTGATGTCGTGATGACGCAGAGCCCTCTCTCTCTTCCCGTTACCCCTGGTGAACCC GCATCAATAAGTTGCCGCTCCAGTCAATCACTTGTACATTCAAATCGCAATACCTACCTG CACTGGTATTTGCAGAAGCCGGGACAATCCCCTCAATTGTTGATATATAAGGTATCCAAT CGCTTTTCTGGAGTTCCTGATAGATTCAGCGGATCCGGGTCTGGTACTGATTTCACTCTG AAAATATCCAGGGTCGAAGCTGAGGACGTAGGCGTATATTATTGCTCTCAGAACACGCAT GTCCCGCCGACTTTCGGCCAGGGCACTAAACTTGAGATCAAGGGTGGGGGGGGCAGCGGT GGTGGAGGCTCTGGTGGAGGAGGGAGCCAGGTCCAACTCGTTCAAAGTGGCGCAGAGGTC AAAAAGCCAGGCGCGAGCGTTAAAGTATCATGTAAGGCCAGCGGTTATACTTTCACTGAT TATGAAATGCACTGGGTGCGACAAGCCCCCGGGCAAGGTCTTGAGTGGATGGGTGCACTT GATCCAAAAACTGGGGATACTGCCTATAGCCAGAAATTCAAAGGGCGCGTCACACTCACT GCCGACAAAAGTACGAGCACAGCTTATATGGAATTGAGTTCACTGACGAGCGAGGATACG GCAGTTTATTACTGTACGCGCTTCTACTCTTACACTTATTGGGGGCAAGGCACTTTGGTT ACTGTGTCCTCTGACAAGACCCATACGTGTCCACCGTGTCCCTTCTGGGTATTGGTTGTG GTCGGCGGTGTCCTTGCTTGTTACAGCCTTCTCGTGACAGTCGCATTCATAATTTTTTGG GTGAAAAGAGGTCGGAAAAAGTTGCTGTATATTTTCAAACAACCCTTTATGAGACCTGTA CAAACGACTCAGGAAGAGGATGGTTGTAGTTGCAGGTTTCCGGAGGAGGAGGAAGGTGGG TGCGAACTGCGGGTGAAATTTAGTAGAAGCGCTGACGCACCAGCTTACCAACAAGGACAG AACCAATTGTACAACGAGCTTAACTTGGGTAGGAGGGAGGAATATGATGTACTGGACAAA AGGCGAGGTCGCGATCCGGAAATGGGAGGCAAGCCACAGCGCCGGAAAAACCCGCAGGAA GGCTTGTACAACGAACTTCAGAAAGATAAAATGGCAGAAGCATACTCCGAAATAGGGATG AAAGGTGAACGGCGGCGAGGCAAGGGCCACGACGGTCTGTACCAAGGGTTGTCAACGGCA ACTAAAGACACGTATGATGCACTTCATATGCAAGCTCTGCCACCCAGGTGA SEQ ID NO: 23 ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAACTGCCCCATCCCGCCTTCCTGCTG ATTCCTGATGTCGTGATGACGCAGAGCCCTCTCTCTCTTCCCGTTACCCCTGGTGAACCC GCATCAATAAGTTGCCGCTCCAGTCAATCACTTGTACATTCAAATCGCAATACCTACCTG CACTGGTATTTGCAGAAGCCGGGACAATCCCCTCAATTGTTGATATATAAGGTATCCAAT CGCTTTTCTGGAGTTCCTGATAGATTCAGCGGATCCGGGTCTGGTACTGATTTCACTCTG AAAATATCCAGGGTCGAAGCTGAGGACGTAGGCGTATATTATTGCTCTCAGAACACGCAT GTCCCGCCGACTTTCGGCCAGGGCACTAAACTTGAGATCAAGGGTGGGGGGGGCAGCGGT GGTGGAGGCTCTGGTGGAGGAGGGAGCCAGGTCCAACTCGTTCAAAGTGGCGCAGAGGTC AAAAAGCCAGGCGCGAGCGTTAAAGTATCATGTAAGGCCAGCGGTTATACTTTCACTGAT TATGAAATGCACTGGGTGCGACAAGCCCCCGGGCAAGGTCTTGAGTGGATGGGTGCACTT GATCCAAAAACTGGGGATACTGCCTATAGCCAGAAATTCAAAGGGCGCGTCACACTCACT GCCGACAAAAGTACGAGCACAGCTTATATGGAATTGAGTTCACTGACGAGCGAGGATACG GCAGTTTATTACTGTACGCGCTTCTACTCTTACACTTATTGGGGGCAAGGCACTTTGGTT ACTGTGTCCTCTGACAAGACCCATACGTGTCCACCGTGTCCCTTCTGGGTATTGGTTGTG GTCGGCGGTGTCCTTGCTTGTTACAGCCTTCTCGTGACAGTCGCATTCATAATTTTTTGG GTGCGGAGCAAGCGGAGCCGGCTGCTGCACTCTGACTACATGAACATGACTCCACGGAGA CCCGGCCCTACCCGGAAACATTATCAGCCCTACGCCCCACCCAGAGATTTTGCCGCTTAT AGGTCCAAAAGAGGTCGGAAAAAGTTGCTGTATATTTTCAAACAACCCTTTATGAGACCT GTACAAACGACTCAGGAAGAGGATGGTTGTAGTTGCAGGTTTCCGGAGGAGGAGGAAGGT GGGTGCGAACTGCGGGTGAAATTTAGTAGAAGCGCTGACGCACCAGCTTACCAACAAGGA CAGAACCAATTGTACAACGAGCTTAACTTGGGTAGGAGGGAGGAATATGATGTACTGGAC AAAAGGCGAGGTCGCGATCCGGAAATGGGAGGCAAGCCACAGCGCCGGAAAAACCCGCAG GAAGGCTTGTACAACGAACTTCAGAAAGATAAAATGGCAGAAGCATACTCCGAAATAGGG ATGAAAGGTGAACGGCGGCGAGGCAAGGGCCACGACGGTCTGTACCAAGGGTTGTCAACG GCAACTAAAGACACGTATGATGCACTTCATATGCAAGCTCTGCCACCCAGGTGA SEQ ID NO: 24 ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAACTGCCCCATCCCGCCTTCCTGCTG ATTCCTCAGGTCCAGCTTGTGCAAAGCGGAGGAGGAGTGGTACAGCCTGGCCGCTCTTTG AGACTGTCTTGTGCGGCCAGTGGATTTACATTCTCTTCTTATGGGTTGCATTGGGTCAGA CAAGCACCGGGCAAAGGATTGGAATGGGTCGCGGCCATTAGCTATGATGGCTCAAAGAAA TATTATGCCGATTCCGTAAAAGGGAGGTTGACAATAAGCCGGGATAACAGCAAGAACACT TTGTATCTTCAGATGAATAGCCTCCGACCGGACGACACGGCACTGTATTTTTGCGCACGC GGGTGGTTTGTAGAACCCCTGAGTTGGGGACAAGGTACTCTTGTCACGGTATCTTCTGGC GGAGGTGGGAGTGGTGGGGGTGGCAGTGGCGGGGGTGGGTCACAAAGCGTGCTTACACAA CCTCCTTCTGCGAGCGGAACTCCGGGACAACGGGTTACGATTTCATGCTCCGGCTCAAGT AGCAATATAGGATCAAATACAGTGAATTGGTATCAACAACTCCCTGGCACAGCGCCCAAG CTGCTGATCTACTCTAATAACCAGAGGCCGAGTGGTGTGCCAGATAGGTTCAGTGGCTCT AAATCAGGTACTAGCGCGAGCCTCGCCATTTCAGGACTTCAATCAGAGGATGAAGCGGAC TACTACTGTGCCGCGTGGGATGATTCACTTAATGGATATGTTTTCGGGACCGGAACAAAA TTGACGGTATTGGAGAGCAAATATGGACCACCATGCCCTCCATGTCCTTTTTGGGTCCTG GTGGTCGTGGGAGGCGTGCTGGCATGTTATTCTCTGCTGGTCACAGTGGCTTTCATCATC TTCTGGGTCAAGCGAGGCCGGAAGAAACTGCTGTACATCTTCAAACAGCCTTTTATGCGC CCAGTGCAGACAACTCAGGAGGAAGACGGCTGCTCTTGTCGGTTCCCCGAGGAAGAGGAA GGGGGATGTGAGCTGCGCGTGAAGTTTTCTCGAAGTGCCGATGCTCCTGCATATCAGCAG GGACAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAATACGACGTGCTG GATAAGAGGCGCGGCAGAGACCCAGAAATGGGCGGGAAGCCACGACGGAAAAACCCCCAG GAGGGGCTGTATAATGAACTGCAGAAGGACAAAATGGCCGAGGCTTACAGCGAAATCGGG ATGAAGGGAGAGAGAAGGCGCGGAAAAGGCCACGATGGACTGTATCAGGGCCTGAGCACT GCCACCAAGGACACCTACGATGCTCTGCACATGCAGGCACTGCCACCCAGGTGA SEQ ID NO: 25 MLLLVTSLLLCELPHPAFLLIPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR QAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR GKRYFDYWGQGTMVTVSSGGGGSGGGGSGGGGSSYELTQPPSASGTPGQRVTISCSGGSS NIGSNTVNWFRQLPGTAPKLLVYFNNQRPSGVPDRFSGSKSGTSASLAIGGLQSDDEADY YCVAWDDSLNAPVFGGGTKVTVLESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIF WVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQG QNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRSEQ ID NO: 26 ATGCTGCTGCTGGTGACAAGCCTGCTGCTGTGCGAACTGCCCCATCCCGCCTTCCTGCTG ATTCCTGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGC CAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACA TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACG CTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGA GGAAAGCGATACTTTGACTACTGGGGCCAGGGGACAATGGTCACCGTCTCGAGTGGTGGG GGGGGCAGCGGTGGTGGAGGCTCTGGTGGAGGAGGGAGCTCCTATGAGCTGACTCAGCCA CCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAGGCAGCTCC AACATCGGAAGTAATACTGTAAACTGGTTCCGGCAGCTCCCAGGAACGGCCCCCAAACTC CTCGTTTATTTTAATAATCAGCGACCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAG TCTGGCACCTCGGCCTCCCTGGCCATCGGTGGGCTCCAGTCTGACGATGAGGCTGACTAT TACTGTGTAGCATGGGATGACTCTCTGAATGCTCCGGTGTTCGGCGGAGGGACCAAGGTC ACCGTCCTAGAGAGCAAATATGGACCACCATGCCCTCCATGTCCTTTTTGGGTCCTGGTG GTCGTGGGAGGCGTGCTGGCATGTTATTCTCTGCTGGTCACAGTGGCTTTCATCATCTTC TGGGTCAAGCGAGGCCGGAAGAAACTGCTGTACATCTTCAAACAGCCTTTTATGCGCCCA GTGCAGACAACTCAGGAGGAAGACGGCTGCTCTTGTCGGTTCCCCGAGGAAGAGGAAGGG GGATGTGAGCTGCGCGTGAAGTTTTCTCGAAGTGCCGATGCTCCTGCATATCAGCAGGGA CAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAATACGACGTGCTGGAT AAGAGGCGCGGCAGAGACCCAGAAATGGGCGGGAAGCCACGACGGAAAAACCCCCAGGAG GGGCTGTATAATGAACTGCAGAAGGACAAAATGGCCGAGGCTTACAGCGAAATCGGGATG AAGGGAGAGAGAAGGCGCGGAAAAGGCCACGATGGACTGTATCAGGGCCTGAGCACTGCC ACCAAGGACACCTACGATGCTCTGCACATGCAGGCACTGCCACCCAGG SEQ ID NO: 27 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKRYFDYWGQGTMVTVSS SEQ ID NO: 28 SYELTQPPSASGTPGQRVTISCSGGSSNIGSNTVNWFRQLPGTAPKLLVYFNNQRPSGVP DRFSGSKSGTSASLAIGGLQSDDEADYYCVAWDDSLNAPVFGGGTKVTVL SEQ ID NO: 29 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY ADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGKRYFDYWGQGTMVTVSS SEQ ID NO: 30 QSVLTQPPSASGTPGQRVTISCSGGSSDIGSNTVNWYQQLPGTAPKLLIYYNNQRPSGVP DRFSGSKSGTSASLAISGLQSEDEADYYCATWDDRMYSPVFGGGTKLTVL SEQ ID NO: 31 DVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNRNTYLHWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIKGGGGSGGG GSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDP KTGDTAYSQKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTV SS SEQ ID NO: 32QVQLVQSGGGVVQPGRSLRLSCAASGFTFS SYGLHWVRQAPGKGLEWVAAISYDGSKKYYADSVKGRLTISRDNSKNTLYLQMNSLRPDD TALYFCARGWFVEPLSWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRV TISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISG LQSEDEADYYCAAWDDSLNGYVFGTGTKLT VLSEQ ID NO: 33 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTC TCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCT CCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTAC GCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT CTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGGAAAG CGATACTTTGACTACTGGGGCCAGGGGACAATGGTCACCGTCTCGAGTGGTGGGGGGGGC AGCGGTGGTGGAGGCTCTGGTGGAGGAGGGAGCTCCTATGAGCTGACTCAGCCACCCTCA GCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAGGCAGCTCCAACATC GGAAGTAATACTGTAAACTGGTTCCGGCAGCTCCCAGGAACGGCCCCCAAACTCCTCGTT TATTTTAATAATCAGCGACCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGC ACCTCGGCCTCCCTGGCCATCGGTGGGCTCCAGTCTGACGATGAGGCTGACTATTACTGT GTAGCATGGGATGACTCTCTGAATGCTCCGGTGTTCGGCGGAGGGACCAAGGTCACCGTC CTA SEQ ID NO: 34GAGGTCCAGCTGCTGGAGAGCGGAGGAGGA CTGGTGCAGCCTGGAGGAAGTCTGCGACTGTCATGCGCCGCTAGCGGCTTCACCTTCAGC TCCTATGCAATGAGCTGGGTGCGACAGGCACCAGGCAAGGGGCTGGAGTGGGTCTCCGCT ATCTCCGGCTCTGGAGGCTCTACTTACTATGCAGACAGTGTGAAGGGGCGGTTCACAATC TCCAGAGATAACTCTAAGAACACTCTGTACCTGCAGATGAACTCTCTGAGAGCTGAGGAC ACCGCAGTGTACTATTGCGCCAAGGGCAAAAGGTACTTTGATTATTGGGGACAGGGCACT ATGGTGACCGTCTCTAGTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGAGGAGGC AGTCAGTCAGTGCTGACACAGCCACCTAGCGCCTCCGGAACCCCAGGACAGCGGGTCACA ATCTCTTGTAGTGGGGGATCAAGCGACATTGGGAGCAACACCGTGAATTGGTATCAGCAG CTGCCTGGAACAGCTCCAAAGCTGCTGATCTACTATAACAATCAGAGGCCCTCCGGCGTC CCTGATCGCTTCTCAGGCAGCAAATCCGGGACTTCTGCAAGTCTGGCCATTAGTGGCCTG CAGTCAGAGGACGAAGCCGATTACTATTGTGCTACCTGGGACGATAGGATGTACTCTCCC GTGTTCGGCGGGGGAACAAAGCTGACTGTC CTGSEQ ID NO: 35 GATGTCGTGATGACGCAGAGCCCTCTCTCTCTTCCCGTTACCCCTGGTGAACCCGCATCA ATAAGTTGCCGCTCCAGTCAATCACTTGTACATTCAAATCGCAATACCTACCTGCACTGG TATTTGCAGAAGCCGGGACAATCCCCTCAATTGTTGATATATAAGGTATCCAATCGCTTT TCTGGAGTTCCTGATAGATTCAGCGGATCCGGGTCTGGTACTGATTTCACTCTGAAAATA TCCAGGGTCGAAGCTGAGGACGTAGGCGTATATTATTGCTCTCAGAACACGCATGTCCCG CCGACTTTCGGCCAGGGCACTAAACTTGAGATCAAGGGTGGGGGGGGCAGCGGTGGTGGA GGCTCTGGTGGAGGAGGGAGCCAGGTCCAACTCGTTCAAAGTGGCGCAGAGGTCAAAAAG CCAGGCGCGAGCGTTAAAGTATCATGTAAGGCCAGCGGTTATACTTTCACTGATTATGAA ATGCACTGGGTGCGACAAGCCCCCGGGCAAGGTCTTGAGTGGATGGGTGCACTTGATCCA AAAACTGGGGATACTGCCTATAGCCAGAAATTCAAAGGGCGCGTCACACTCACTGCCGAC AAAAGTACGAGCACAGCTTATATGGAATTGAGTTCACTGACGAGCGAGGATACGGCAGTT TATTACTGTACGCGCTTCTACTCTTACACTTATTGGGGGCAAGGCACTTTGGTTACTGTG TCCTCT SEQ ID NO: 36CAGGTCCAGCTTGTGCAAAGCGGAGGAGGA GTGGTACAGCCTGGCCGCTCTTTGAGACTGTCTTGTGCGGCCAGTGGATTTACATTCTCT TCTTATGGGTTGCATTGGGTCAGACAAGCACCGGGCAAAGGATTGGAATGGGTCGCGGCC ATTAGCTATGATGGCTCAAAGAAATATTATGCCGATTCCGTAAAAGGGAGGTTGACAATA AGCCGGGATAACAGCAAGAACACTTTGTATCTTCAGATGAATAGCCTCCGACCGGACGAC ACGGCACTGTATTTTTGCGCACGCGGGTGGTTTGTAGAACCCCTGAGTTGGGGACAAGGT ACTCTTGTCACGGTATCTTCTGGCGGAGGTGGGAGTGGTGGGGGTGGCAGTGGCGGGGGT GGGTCACAAAGCGTGCTTACACAACCTCCTTCTGCGAGCGGAACTCCGGGACAACGGGTT ACGATTTCATGCTCCGGCTCAAGTAGCAATATAGGATCAAATACAGTGAATTGGTATCAA CAACTCCCTGGCACAGCGCCCAAGCTGCTGATCTACTCTAATAACCAGAGGCCGAGTGGT GTGCCAGATAGGTTCAGTGGCTCTAAATCAGGTACTAGCGCGAGCCTCGCCATTTCAGGA CTTCAATCAGAGGATGAAGCGGACTACTACTGTGCCGCGTGGGATGATTCACTTAATGGA TATGTTTTCGGGACCGGAACAAAATTGACG GTATTGSEQ ID NO: 37 GFTFSSYAMS SEQ ID NO: 38 AISGSGGSTYYADSVKG SEQ ID NO: 39GKRYFDY SEQ ID NO: 40 SGGSSNIGSNTVN SEQ ID NO: 41 FNNQRPS SEQ ID NO: 42VAWDDSLNAPV SEQ ID NO: 43 SGGSSDIGSNTVN SEQ ID NO: 44 YNNQRPSSEQ ID NO: 45 ATWDDRMYSPV SEQ ID NO: 46 ATGCGGAGCAAAGAAAGCGAGGTGTTCTACGAGCTGGCCCACCAACTGCCTCTGCCTCAC AATGTGTCCAGCCACCTGGATAAGGCCAGCGTGATGAGACTGACCATCAGCTACCTGAGA GTGCGGAAGCTGCTGGATGCCGGCGATCTGGACATCGAGGACGATATGAAGGCCCAGATG AACTGCTTCTACCTGAAGGCCCTGGACGGCTTCGTGATGGTGCTGACCGATGACGGCGAC ATGATCTACATCAGCGACAACGTGAACAAGTACATGGGGCTGACCCAGTTCGAGCTGACA GGCCACAGCGTGTTCGACTTCACACACCCCTGCGACCACGAAGAGATGAGAGAGATGCTG ACCCACCGGAACGGCCTGGTCAAGAAGGGCAAAGAGCAGAATACCCAGCGGTCATTCTTC CTGCGGATGAAGTGCACCCTGACCAGCAGGGGCAGAACCATGAACATCAAGAGCGCCACA TGGAAGGTGCTGCACTGCACCGGACACATCCACGTGTACGACACCAACAGCAACCAGCCT CAGTGCGGCTACAAGAAACCTCCTATGACCTGCCTGGTGCTGATCTGCGAGCCCATTCCT CATCCTAGCAACATCGAGATCCCTCTGGACAGCAAGACCTTCCTGAGCAGACACAGCCTG GACATGAAGTTCAGCTACTGCGACGAGCGGATCACCGAGCTGATGGGCTATGAGCCTGAA GAACTGCTGGGCCGCAGCATCTACGAGTACTATCACGCCCTGGACAGCGACCACCTGACC AAGACACACCACGACATGTTCACCAAGGGCCAAGTGACCACCGGCCAGTACAGAATGCTG GCCAAGCGCGGAGGCTACGTGTGGGTTGAAACACAGGCCACCGTGATCTACAACACCAAG AACTCCCAGCCACAGTGCATCGTGTGCGTGAACTACGTGGTGTCCGGCATCATCCAGCAC GACCTGATCTTCAGCCTGCAGCAGACCGAGTGCGTGCTGAAGCCTGTGGAAAGCAGCGAC ATGAAGATGACCCAGCTGTTTACCAAGGTGGAATCCGAGGACACCAGCAGCCTGTTCGAC AAG SEQ ID NO: 47MRSKESEVFYELAHQLPLPHNVSSHLDKAS VMRLTISYLRVRKLLDAGDLDIEDDMKAQMNCFYLKALDGFVMVLTDDGDMIYISDNVNK YMGLTQFELTGHSVFDFTHPCDHEEMREMLTHRNGLVKKGKEQNTQRSFFLRMKCTLTSR GRTMNIKSATWKVLHCTGHIHVYDTNSNQPQCGYKKPPMTCLVLICEPIPHPSNIEIPLD SKTFLSRHSLDMKFSYCDERITELMGYEPEELLGRSIYEYYHALDSDHLTKTHHDMFTKG QVTTGQYRMLAKRGGYVWVETQATVIYNTKNSQPQCIVCVNYVVSGIIQHDLIFSLQQTE CVLKPVESSDMKMTQLFTKVESEDTSSLFDKIYNTKNSQPQCIVCVNYVVSGIIQHDLIF SLQQTECVLKPVESSDMKMTQLFTKVESED TSSLFDK

What is claimed is:
 1. An isolated nucleic acid sequence encoding a) achimeric antigen receptor (CAR), wherein the CAR comprises anantigen-binding domain specific for a cell surface antigen; and b) anarmoring molecule, wherein the armoring molecule countersimmunosuppression of a cell in a tumor microenvironment when expressedon a surface of the cell.
 2. The isolated nucleic acid sequence of claim1, wherein the antigen-binding domain comprises an antibody orantigen-binding fragment thereof.
 3. The isolated nucleic acid sequenceof claim 2, wherein the antigen-binding domain is a Fab or a singlechain variable fragment (scFv).
 4. The isolated nucleic acid sequence ofclaim 3, wherein the antigen-binding domain is an scFv comprising thenucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO:
 34. 5. The isolatednucleic acid sequence according to any of the preceding claims furtherencoding a transmembrane domain, a costimulatory domain, and a signaldomain.
 6. The isolated nucleic acid sequence of claim 5, wherein thetransmembrane domain comprises a CD28 transmembrane domain.
 7. Theisolated nucleic acid sequence of claim 5, wherein the costimulatorydomain comprises one or more of CD28, 4-1BB, CD3zeta, OX-40, ICOS, CD27,GITR, and MyD88/CD40 costimulatory domains.
 8. The isolated nucleic acidsequence of claim 5, wherein the costimulatory domain comprises one ormore of CD28, 4-1BB, and CD3zeta costimulatory domains.
 9. The isolatednucleic acid sequence of claim 5, wherein the signal domain comprises asequence encoding a CSFR2 signal peptide.
 10. The isolated nucleic acidsequence according to any of the preceding claims a sequence encodingfurther comprising a hinge/spacer domain.
 11. The isolated nucleic acidsequence of claim 10, wherein the hinge/spacer domain is an IgG4Phinge/spacer.
 12. The isolated nucleic acid sequence of claim 1, whereinthe armoring molecule is hypoxia-inducible factor 1α(HIF-1α) dominantnegative (HIF 1 αDN).
 13. The isolated nucleic acid sequence of claim12, wherein HIF1αDN is encoded by the nucleic acid sequence of SEQ IDNO:
 46. 14. The isolated nucleic acid of claim 1, wherein the cellsurface antigen comprises one or more of CD10, CD16, CD19, CD20, CD22,CD123, CD30, CD34, CD47, CD56, CD80, CD86, CD117, CD133, CD138, CD171,CD37, CD38, CD5, CD7, CD79, 5T4, AFP, AXL, BCMA, B7H3, CDH3, CDH6,CLDN6, CLDN18, CLL-1, CMV, CS1, DLL3, DR5, FBP, GD2, GFRA1, GPA33, GPC3,IL-1-RAP, IL17RA, ITGB7, EBV, ERBB1/EGFR, ERBB2/Her-2, ERBB3, ERBB4,cMet, EGFR vIII, FAP, FOLR1, CEA, CEACAM6, EphA2, HSV-1, HSV-2, HTLV,HPV16-E6, HPV16-E7, IL13Ra2, IgK chain, LGR5, LMP1, LeY, LRP8, MG7, MR1,NRCAM, PMEL, NKG2D ligand, PRAME, PRLR, PVR, ROR1, ROR2, SSX2, STEAPI,STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2, MAGE-A1,MAGE-A3, MAGE-A4, mesothelin (MSLN), MUC1, MUC16, NY-ESO-1, WT1, PDL1,CAIX, CD70, PSMA, and PSCA.
 15. A vector, comprising a nucleic acidsequence encoding a chimeric antigen receptor (CAR), wherein the CARcomprises an antigen-binding domain specific for a cell surface antigenand an armoring molecule, wherein the nucleic acid sequence comprisesSEQ ID NO:
 46. 16. A cell comprising the vector of claim 15 or theisolated nucleic acid of any of claims 1-14.
 17. A cell, comprising: anucleic acid sequence encoding a chimeric antigen receptor (CAR), and aHIF1αDN armoring molecule expressed on a surface of the cell.
 18. Thecell of claim 17, wherein the CAR comprises an antigen-binding domain, atransmembrane domain, a costimulatory domain, and a signal domain. 19.The cell of claim 18, wherein the antigen-binding domain is a Fab or asingle chain variable fragment (scFv).
 20. A cell, comprising: ananti-GPC3 chimeric antigen receptor (CAR) comprising an antigen bindingdomain, wherein the antigen binding domain comprises an antibody, Fab,or an scFv comprising a heavy chain variable region (VH) and a lightchain variable region (VL), wherein the VH comprises a CDR1 comprisingthe amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the aminoacid sequence of SEQ ID NO: 38, and a CDR3 comprising the amino acidsequence of SEQ ID NO: 39, and wherein the VL comprises a CDR1comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 43, aCDR2 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO:44, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 42 orSEQ ID NO: 45; and a HIF1αDN armoring molecule.
 21. The cell of claim20, wherein the VH comprises the amino acid sequence of SEQ ID NO: 27 orSEQ ID NO:
 29. 22. The cell of claim 20-21, wherein the VL comprises theamino acid sequence of SEQ ID NO: 28 or SEQ ID NO:
 30. 23. The cell ofany of claims 20-22, wherein the HIF1αDN armoring molecule comprises theamino acid sequence of SEQ ID NO:
 47. 24. The cell of any of claims16-23, wherein the cell is selected from the group consisting of a Tcell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), and aregulatory T cell.
 25. A method of treating cancer, comprising:administering to a subject in need thereof a cell, wherein the cellcomprises a) a chimeric antigen receptor (CAR) specific for a cellsurface antigen, and b) an armoring molecule, wherein the armoringmolecule counters immunosuppression of the cell in a tumormicroenvironment of the cancer.
 26. The method of claim 25, wherein thecell surface antigen is one or more of CD10, CD16, CD19, CD20, CD22,CD123, CD30, CD34, CD47, CD56, CD80, CD86, CD117, CD133, CD138, CD171,CD37, CD38, CD5, CD7, CD79, 5T4, AFP, AXL, BCMA, B7H3, CDH3, CDH6,CLDN6, CLDN18, CLL-1, CMV, CS1, DLL3, DR5, FBP, GD2, GFRA1, GPA33, GPC3,IL-1-RAP, IL17RA, ITGB7, EBV, ERBB1/EGFR, ERBB2/Her-2, ERBB3, ERBB4,cMet, EGFRvIII, FAP, FOLR1, CEA, CEACAM6, EphA2, HSV-1, HSV-2, HTLV,HPV16-E6, HPV16-E7, IL13Ra2, IgK chain, LGR5, LMP1, LeY, LRP8, MG7, MR1,NRCAM, PMEL, NKG2D ligand, PRAME, PRLR, PVR, ROR1, ROR2, SSX2, STEAPI,STEAP2, TACI, TIM3, TRBC1, VEGFR-2, EPCAM1, VCAM1, VIPR2, MAGE-A1,MAGE-A3, MAGE-A4, mesothelin (MSLN), MUC1, MUC16, NY-ESO-1, WT1, PDL1,CAIX, CD70, PSMA, and PSCA.
 27. The method of claim 26, wherein thearmoring molecule a HIF1caDN armoring molecule.
 28. The method of claim27 further comprising inhibiting tumor growth, inducing tumorregression, and/or prolonging survival of the subject.
 29. The method ofclaim 25, wherein the cell is an autologous cell.
 30. The method ofclaim 29, wherein the autologous cell is selected from the groupconsisting of a T cell, a Natural Killer (NK) cell, a cytotoxic Tlymphocyte (CTL), and a regulatory T cell.
 31. The method of any ofclaims 25-30, wherein the cancer is a solid tumor.
 32. The method ofclaim 31, wherein the cancer is hepatocellular carcinoma, non-small celllung cancer, ovarian cancer, and/or squamous cell lung carcinoma. 33.The method of claim 32, wherein the cancer is hepatocellular carcinoma.34. The method of any of claims 26-33 further comprising administeringto the subject a therapeutically effective amount of an anticancerantibody and/or a chemotherapeutic component.